Lactiplantibacillus plantarum AUY2301 with antibacterial, immunomodulatory and oxidative damage alleviating effects and its application

Abstract

The present invention provides Lactiplantibacillus plantarum AUY2301 with antibacterial, immunomodulatory and oxidative damage alleviating effects and its application. The Lactiplantibacillus plantarum strain AUY2301 is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 30125. The present invention discovers through experiments that the strain AUY2301 has functions of strong intestinal and gastric juice tolerance, antibacterial property, good adhesiveness, immunomodulatory activity, anti-inflammatory property and oxidative damage alleviating effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to Lactiplantibacillus plantarum AUY2301 with antibacterial, immunomodulatory and oxidative damage-relieving effects and its applications. Background Art

[0002] Modern people may experience a decline in body immunity due to physical fatigue, lack of exercise, work pressure, irregular diet and rest, etc., showing a sub-healthy state. Low immunity makes the body vulnerable to the invasion of harmful substances such as pathogenic bacteria, leading to colds and other diseases. The immune system is crucial for the body to defend against pathogens, but the immune system is also affected by many factors such as unhealthy living habits, pathogens and antigens. Immunomodulators are a class of substances that change the innate and acquired immune systems by increasing, decreasing or modifying the body's immune response, but the use of this series of drugs is limited by their toxic side effects and high costs. At the same time, most immunomodulatory drugs are not suitable for use as prophylactic drugs. An effective way to improve immunity is to pay attention to a balanced diet, reasonably match various nutrients, and intake nutritional health products such as probiotics and fermented dairy products.

[0003] Lactiplantibacillus plantarum is an important component of the body's innate immune system and an indispensable part for the body to maintain the stable state of immune function. It plays a crucial role in maintaining the host microecological balance and improving the body's immune system function, and its use value has been increasingly emphasized by people. Summary of the Invention

[0004] In view of this, the present invention provides Lactiplantibacillus plantarum AUY2301 with antibacterial, immunomodulatory and oxidative damage-relieving effects and its applications. The Lactiplantibacillus plantarum AUY2301 has the functions of being resistant to intestinal juice, gastric juice, antibacterial, regulating immune activity, anti-inflammatory and relieving oxidative damage.

[0005] To solve the technical problems raised in the background art, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a strain of Lactiplantibacillus plantarum AUY2301, which is deposited in the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No. 30125.

[0007] In the second aspect, the present invention provides a microbial preparation, including the strain of Lactiplantibacillus plantarum AUY2301 as described above.

[0008] In a third aspect, the present invention provides the use of the Lactiplantibacillus plantarum strain AUY2301 as described above or the microbial preparation as described above in the preparation of a medicament having the following effects:

[0009] Intestinal fluid tolerance; and / or

[0010] Gastric juice tolerance; and / or

[0011] Cell adhesion; and / or

[0012] Bacteriostasis; and / or

[0013] Regulating immune activity; and / or

[0014] Anti-inflammatory; and / or

[0015] Ability to relieve oxidative damage.

[0016] Furthermore, the cell adhesion includes adhesion to intestinal epithelial Caco-2 cells.

[0017] Furthermore, the bacteriostasis includes inhibiting Listeria and Escherichia coli.

[0018] Furthermore, the regulation of immune activity includes promoting the release of immune cytokines by cells.

[0019] Furthermore, the anti-inflammatory includes inhibiting the release of inflammatory factors.

[0020] Furthermore, the ability to relieve oxidative damage includes: increasing the lifespan, reproduction, and motility of nematodes modeled with oxidative damage, and inhibiting the release of superoxide dismutase SOD, catalase CAT, reactive oxygen species ROS, and malondialdehyde MDA in nematodes modeled with oxidative damage.

[0021] Optionally, the promoting the release of immune cytokines by cells refers to promoting the release of immune cytokines by healthy cells, and the immune cytokines include at least one of IL-6, IL-1β, TNF-α, and NO.

[0022] Optionally, the inhibiting the release of inflammatory factors refers to inhibiting the release of inflammatory factors in inflammation-modeled cells, and the inflammatory factors include at least one of IL-6, IL-1β, TNF-α, NO, and PGE2.

[0023] In a fourth aspect, the present invention provides a fermented product, which is prepared by fermenting with the Lactiplantibacillus plantarum strain AUY2301 as described above or the microbial preparation as described above.

[0024] Fifth aspect, the present invention provides a food, which comprises at least one of the above-mentioned Lactiplantibacillus plantarum strain AUY2301, the above-mentioned microbial preparation, and the above-mentioned fermented product; and acceptable excipients.

[0025] Sixth aspect, the present invention provides a medicine, which comprises at least one of the above-mentioned Lactiplantibacillus plantarum strain AUY2301, the above-mentioned microbial preparation, and the above-mentioned fermented product; and acceptable excipients.

[0026] Seventh aspect, the present invention provides a nutritional health product, which comprises at least one of the above-mentioned AUY2301, the above-mentioned microbial preparation, and the above-mentioned fermented product; and acceptable excipients.

[0027] The beneficial effects of the above technical solutions of the present invention are as follows:

[0028] The present invention provides a Lactiplantibacillus plantarum strain AUY2301, which is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 30125.

[0029] The Lactiplantibacillus plantarum AUY2301 provided in the present invention has at least the following properties:

[0030] (1) AUY2301 has good ability to tolerate simulated gastric juice. In simulated gastric juice, after incubation for 3 h, the survival rate can reach 98.03%.

[0031] (2) AUY2301 has good ability to tolerate simulated intestinal fluid. After incubation in simulated intestinal fluid for 3 h, the survival rate can reach 102.96%.

[0032] (3) AUY230 has good adhesion performance. The adhesion ability reaches 30 bacteria per cell after incubation with Caco-2 cells for 2 h.

[0033] (4) AUY2301 has good antibacterial activity.

[0034] (5) AUY2301 can promote the proliferation of RAW264.7 cells; can promote the phagocytic activity of RAW264.7 cells; can significantly increase the release of immune cytokines IL-6, IL-1β, TNF-α and NO in RAW264.7 cells; can significantly inhibit the expression of COX-2 protein in RAW264.7 induced by LPS; can significantly inhibit the release of PGE2 in RAW264.7 induced by LPS; AUY2301 can significantly inhibit the release of inflammatory factors IL-6, IL-1β, TNF-α and NO in RAW264.7 induced by LPS through the NF-κB signaling pathway, indicating that AUY2301 has the functions of regulating immune activity and anti-inflammation;

[0035] (6) AUY2301 can promote the lifespan, reproductive ability and motility of Caenorhabditis elegans with oxidative damage induced by LPS, and inhibit the release of SOD, CAT, ROS and the release of MDA in the nematodes with oxidative damage model, indicating that AUY2301 has the function of alleviating oxidative damage.

[0036] Biomaterial Deposit

[0037] Biomaterial: Strain AUY2301, classified and named: Lactiplantibacillus plantarum, was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 25, 2024, at the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 30125. Description of the Drawings

[0038] Figure 1 Effect of AUY2301 on COX-2 in RAW264.7 macrophages induced by LPS;

[0039] Figure 2 Effect of AUY2301 on the expression of proteins in the NF-κB signaling pathway;

[0040] Figure 3 Effect of different concentrations of LPS on the survival rate of nematodes;

[0041] Figure 4 Effect of AUY2301 on the lifespan of nematodes under oxidative stress induced by LPS;

[0042] Figure 5 Effect of AUY2301 on the reproductive ability of nematodes under oxidative stress induced by LPS;

[0043] Figure 6 Effect of AUY2301 on the motility of nematodes under oxidative stress induced by LPS. Specific embodiments

[0044] The present invention discloses a Lactiplantibacillus plantarum strain AUY2301 and its applications. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0045] In a first aspect, the present invention provides a Lactiplantibacillus plantarum strain AUY2301, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 30125.

[0046] The present invention provides a strain AUY2301, the taxonomic naming of the strain AUY2301 is Lactiplantibacillus plantarum, and it was deposited in the China General Microbiological Culture Collection Center on March 25, 2024. The address is: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 30125.

[0047] In a second aspect, the present invention provides a microbial preparation, which includes the above-mentioned Lactiplantibacillus plantarum strain AUY2301.

[0048] In a third aspect, the present invention provides the application of the above-mentioned Lactiplantibacillus plantarum strain AUY2301 or the above-mentioned microbial preparation in the preparation of a drug having the following effects:

[0049] Intestinal juice tolerance; and / or

[0050] Gastric juice tolerance; and / or

[0051] Cell adhesion; and / or

[0052] Bacteriostasis; and / or

[0053] Regulating immune activity; and / or

[0054] Anti-inflammatory; and / or

[0055] Ability to relieve oxidative damage.

[0056] According to some embodiments of the present invention, the product is a drug.

[0057] According to some embodiments of the present invention, the Lactiplantibacillus plantarum strain AUY2301 showed tolerance to intestinal fluid in the experiment of simulated intestinal fluid. The formula of the simulated intestinal fluid is as follows: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, adjust the pH value to 6.8 with 0.4% NaOH solution; separately take 10 g of trypsin, add an appropriate amount of water to dissolve it, mix the two solutions, then dilute with water to 1000 mL, and filter and sterilize with a microporous membrane with a pore size of 0.20 μm to prepare artificial intestinal fluid for standby.

[0058] According to some embodiments of the present invention, the Lactiplantibacillus plantarum strain AUY2301 showed tolerance to gastric fluid in the experiment of simulated gastric fluid. The formula of the simulated gastric fluid is as follows: Prepare a solution with a pH of 3. Measure 20 mL of 1 mol / L HCl respectively, and then adjust the pH value to 3 with 0.4% NaOH solution; add pepsin and dissolve it to make its mass concentration 1 g / 100 mL, and sterilize with a 0.22 μm microporous membrane to prepare artificial gastric fluid for standby.

[0059] Further, the cell adhesion includes, but is not limited to, the adhesion to intestinal epithelial cells Caco-2 cells. The strain AUY2301 in the present invention has good cell adhesion. The cell adhesion of the strain is crucial for the survival, adaptation and interaction of microorganisms in the environment, and is also of great significance for bioengineering applications.

[0060] Further, the antibacterial activity includes, but is not limited to, inhibiting Listeria and Escherichia coli.

[0061] Further, the regulation of immune activity includes promoting the release of immune cytokines by cells.

[0062] According to some embodiments of the present invention, the promotion of the release of immune cytokines by cells means promoting the release of immune cytokines by healthy cells, and the immune cytokines include at least one of IL-6, IL-1β, TNF-α and NO.

[0063] Further, the anti-inflammatory effect includes inhibiting the release of inflammatory factors.

[0064] According to some embodiments of the present invention, the inhibition of the release of inflammatory factors means inhibiting the release of inflammatory factors in inflammation-induced cells, and the inflammatory factors include at least one of IL-6, IL-1β, TNF-α, NO and PGE2.

[0065] Furthermore, the ability to alleviate oxidative damage includes: increasing the lifespan, reproduction, and motility of nematodes with oxidative damage-induced models, as well as inhibiting the release of SOD, CAT, and ROS and the release of MDA in nematodes with oxidative damage-induced models.

[0066] Fourthly, the present invention provides a fermented product, which is prepared by fermenting with the Lactiplantibacillus plantarum strain AUY2301 as described above or the microbial preparation as described above.

[0067] Fifthly, the present invention provides a food, which includes at least one of the Lactiplantibacillus plantarum strain AUY2301 as described above, the microbial preparation as described above, and the fermented product as described above; and acceptable excipients.

[0068] Sixthly, the present invention provides a medicine, which includes at least one of the Lactiplantibacillus plantarum strain AUY2301 as described above, the microbial preparation as described above, and the fermented product as described above; and acceptable excipients.

[0069] Seventhly, the present invention provides a nutritional health product, which includes at least one of AUY2301 as described above, the microbial preparation as described above, and the fermented product as described above; and acceptable excipients.

[0070] Screening of strain AUY2301:

[0071] The present invention uses the CCK-8 method to screen lactic acid bacteria with immunomodulatory activity from a strain library including 84 lactic acid bacteria strains. The CCK-8 method is carried out according to the operation manual of the CCK-8 kit, and the CCK-8 kit is purchased from Beyotime Biotechnology Co., Ltd.

[0072] The sources of the strains in the strain library are as follows: Dozens of healthy milk samples of different varieties of cattle and sheep are collected from pastures in different southern provinces of China. The samples are serially diluted by 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 , 200 μL is taken from each dilution and spread on an MRS solid plate (containing 2% CaCO 3 ), each sample is replicated three times, and then the plate is placed in a 37 °C incubator for 24 - 48 h. Single colonies with obvious calcium dissolution rings and typical lactic acid bacteria morphology are selected, purified and then inoculated into MRS broth medium, and stored in a 4 °C refrigerator for later use.

[0073] The present invention uses the CCK-8 method to screen lactic acid bacteria with immunomodulatory activity from 84 strains of lactic acid bacteria. Among them, AUY2301 can promote the proliferation of immune cells; the neutral red experiment verifies that strain AUY2301 can improve the phagocytic activity of macrophages: further ELISA experiments find that AUY2301 can not only increase the release of cytokines in RAW264.7 cells but also inhibit the excessive release of inflammatory factors and inflammatory mediators in LPS-induced RAW264.7 cells. In Caenorhabditis elegans, AUY2301 can increase the lifespan, reproduction, motility of nematodes with oxidative damage models, and inhibit the release of SOD, CAT, ROS and the release of MDA in nematodes with oxidative damage models; the viable bacteria of AUY2301 still have antibacterial effects on indicator bacteria.

[0074] Through morphological observation and 16S rRNA gene sequence analysis, strain AUY2301 was identified as Lactiplantibacillus plantarum.

[0075] The 16S rRNA gene sequence of strain AUY2301 is shown in SEQ ID No.1. When this sequence was aligned with nucleic acid sequences in NCBI, the results showed that the strain was Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum AUY2301.

[0076] The following further illustrates the present invention through some specific examples.

[0077] Unless otherwise specified, the raw materials and reagents used in the following examples can be purchased from the market.

[0078] Example 1 Tolerance experiment of Lactiplantibacillus plantarum AUY2301 to simulated gastrointestinal fluids

[0079] Preparation of artificial gastric juice: Prepare a solution with a pH of 3. Measure 20 mL of 1 mol / L HCl respectively, and then add 0.4% NaOH solution to adjust the pH value to 3; add pepsin and dissolve it to make its mass concentration 1 g / 100 mL, and sterilize it with a microporous filter membrane with a pore size of 0.22 μm to obtain artificial gastric juice for standby.

[0080] Preparation of artificial intestinal juice: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, and add 0.4% NaOH solution to adjust the pH value to 6.8; take another 10 g of trypsin, add an appropriate amount of water to dissolve it, mix the two solutions, and then dilute with water to 1000 mL, and filter and sterilize it with a microporous filter membrane with a pore size of 0.20 μm to obtain artificial intestinal juice for standby.

[0081] Activate Lactobacillus AUY2301 and prepare a bacterial suspension with a viable bacteria count of 1×10 8CFU / mL, and then add 0.5 mL of the bacterial suspension to 4.5 mL of artificial gastric juice and artificial intestinal juice respectively. Vortex mix for 15 s, incubate in an environment of 37 °C for 3 h, and use the plate counting method to measure the viable bacteria count at 0 h and 3 h respectively. The survival rate of lactic acid bacteria in gastrointestinal juice is calculated as follows:

[0082] Survival rate calculation formula = viable bacteria count at 3 h / viable bacteria count at 0 h * 100%.

[0083] Result analysis:

[0084] Table 1 records the data of the tolerance ability of Lactiplantibacillus plantarum AUY2301 to simulated gastric juice. From the survival rate in Table 1, it can be seen that after the strain AUY2301 was incubated in simulated gastric juice for 2 h, the survival rate could still reach 98.03%, indicating that the strain AUY2301 has good acid tolerance ability.

[0085] Table 1

[0086]

[0087] Table 2 records the data of the tolerance ability of Lactiplantibacillus plantarum AUY2301 to simulated intestinal juice. From the survival rate in Table 2, it can be seen that after the strain AUY2301 was cultured in simulated intestinal juice for 3 h, the survival rate reached 102.96%, indicating that the strain AUY2301 has good tolerance ability to intestinal juice.

[0088] Table 2

[0089]

[0090] Example 2 Antibacterial experiment of Lactiplantibacillus plantarum AUY2301

[0091] First, dilute the bacterial suspensions of Escherichia coli (ATCC33760) and Listeria monocytogenes (purchased from Qingdao Haibo Biotechnology Co., Ltd.) cultured for 24 h after activation with sterile normal saline to a concentration of 10 5 CFU / mL, and then coat it on the BHI solid medium with a sterile cotton swab. After the bacterial liquid is dried, place the Oxford cup on the plate. Culture the test Lactobacillus AUY2301 for 24 h, centrifuge at 8000 RPM for 10 min, take 200 μL of the supernatant, add it to the Oxford cup on the indicator plate, and let the plate stand in a 4 °C refrigerator for 4 - 6 h. After the supernatant has completely diffused into the agar, transfer it to a 37 °C incubator. Set 3 replicates for each strain, and use an equal amount of MRS liquid blank medium for the control wells. After culturing for 12 h, take the plate out of the 37 °C incubator, remove the Oxford cup with sterile forceps, and measure the antibacterial diameter with a vernier caliper. Select the strain with an antibacterial circle diameter ≥ 13 mm as the standard.

[0092] Result analysis:

[0093] Table 3 records the diameters of the inhibition zones of strain AUY2301 against the above two indicator bacteria, indicating that strain AUY2301 has good effects in inhibiting Escherichia coli and Listeria.

[0094] Table 3

[0095]

[0096] Example 3: Test on the cell adhesion ability of Lactiplantibacillus plantarum AUY2301

[0097] Adhesion to Caco-2 cells:

[0098] Cell culture: Take the Caco-2 cells stored in liquid nitrogen and quickly thaw them in water at 37°C. Centrifuge at 800 r / min for 5 min, add 1 mL of high-glucose DMEM medium and mix well. Transfer it into a cell culture flask, and culture it in a DMEM medium containing 10% fetal bovine serum (FBS) in a 5% CO 2 , 37°C incubator. Change the medium every two days. When the cells grow to more than 80% of the culture flask, passage the cells. After passage 3 times, use them for the experiment.

[0099] Adhesion test: Add 1 mL of Caco-2 cell suspension (1.0x10 5 cells / mL) into a 6-well cell culture plate, and culture it in a CO 2 incubator until the cells grow to a monolayer. Aspirate the culture medium, wash it twice with sterile PBS, add 1 mL of bacterial suspension (here, the bacterial suspension of Lactobacillus AUY2301 is resuspended with a DMEM medium containing 10% FBS), and incubate it in the incubator for 2 h; then aspirate the bacterial suspension, wash it 3 times with sterile PBS to wash away the non-adherent bacteria. Finally, collect the cells with a cell scraper, and use a hemocytometer to measure the number of Caco-2 cells in each well and the plate counting method to measure the number of adherent bacteria.

[0100] Result analysis: Table 4 records that the number of adherent cells of strain AUY2301 to Caco-2 cells within 2 h is 30 cfu / cell.

[0101] Table 4

[0102] Strain Number of adherent cells (cfu / cell) Lactiplantibacillus plantarum AUY2301 30

[0103] Example 4: Experiment on the effect of strain AUY2301 on macrophage proliferation

[0104] Preparation of bacterial suspension: Inoculate strain AUY2301 into MRS medium, incubate at 37 °C for 24 h, subculture and activate three times, centrifuge at 4 °C and 10000 r / min for 15 min, remove the supernatant, wash the bacterial sludge with physiological saline, dilute it, and adjust the bacterial suspension to 0.8 using an enzyme-linked immunosorbent assay (ELISA) reader to obtain a bacterial suspension, which is prepared and used immediately. The commercial strain Lactobacillus rhamnosus GG (LGG) is used as the positive control group.

[0105] Culture and treatment of macrophages: RAW264.7 cells are cultured in a medium containing 10% fetal bovine serum (FBS) + high-glucose DMEM at 37 °C and 5% CO 2 conditions. When digesting, remove the medium, add 2 mL of PBS to wash and then discard it. Subsequently, add 1 mL of trypsin, aspirate after 60 s of digestion, add an appropriate amount of medium, blow the cells off the wall to form a suspension, and transfer the suspension to a new container. After the cells stably cover the cell flask, transfer them to a suitable well plate and grow until 90%.

[0106] Using the CCK-8 method, inoculate 200 μL of cells in the logarithmic growth phase into a 96-well plate, with 2×10 4 cells in each well, and place them in an incubator at 37 °C for pre-incubation for 24 hours. Remove the old medium in the 96-well plate and replace it with fresh serum-free medium, and place it in an incubator at 37 °C for pre-incubation for 2.5 h. Add the bacterial suspension with an OD 600nm value of 0.8 to the 96-well plate and continue to culture in an incubator at 37 °C for 12 h. According to the method provided by the CCK-8 kit (Beyotime Biotechnology Co., Ltd.), after the reaction, add 20 μL of CCK-8 solution to each well and treat for 3 h. Use an ELISA reader to measure the absorbance of each sample at 450 nm and calculate the survival rate.

[0107] Results and analysis

[0108] The results of strain AUY2301 promoting the proliferation of immune cells are shown in Table 5. As shown in Table 5, compared with the blank control group, AUY2301 significantly improved the proliferation ability of RAW264.7 macrophages at an optical density of 0.8 (p < 0.05). At the same time, AUY2301 had no cytotoxicity to RAW264.7 macrophages when the OD 600nm value was 0.8.

[0109] Table 5

[0110]

[0111] Note: The results in the table are averaged ± standard deviation after three parallel experiments.

[0112] Effect of Strain AUY2301 on Phagocytic Activity of Immune Cells

[0113] A negative control group (only cells), a positive control group (commercial strain Lactobacillus rhamnosus (LGG) + cells), and an experimental group (strain AUY2301 + cells) were set up. Macrophage RAW264.7 was treated according to the method in Example 4. After culturing for 12 h, 100 μL of 0.072% neutral red solution was added to each well, and after continuing to culture for 30 min, the neutral red was discarded. The cells were washed twice with PBS, and 100 μL of cell lysate (ethanol∶acetic acid = 1∶1, V / V) was added to each well and placed in a refrigerator at 4°C overnight. After the cells were completely lysed, the optical density value at a wavelength of 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the phagocytic activity was calculated. The calculation formula is as follows:

[0114] Phagocytic activity = As / Ac × 100%;

[0115] In the formula: As is the OD 540nm value of the negative control group; Ac is the OD 540nm value of the experimental group.

[0116] As shown in Table 6, compared with the control group, when cells were treated with AUY2301, the phagocytic activity was 1.2 times higher than that of the blank control group and higher than that of strain LGG, indicating that strain AUY2301 can improve the phagocytic activity of macrophages.

[0117] Table 6

[0118]

[0119] Example 6 Effect of Strain AUY2301 on the Release of NO, IL-6, IL-1β, and TNF-α in Macrophages

[0120] Macrophage RAW264.7 was inoculated into a 24-well plate, with 1 mL of cell suspension (cell concentration of 5×10 5 cells / mL) in each well, and cultured at 37°C and 5% CO 2 for 24 h. The cells adhered and grew. The culture medium was discarded. A negative control group (only cells), a positive control group (LGG + cells), and an experimental group (strain AUY2301 + cells) were set up, with 6 replicate wells in each group, and cultured at 37°C and 5% CO 2 for 12 h. The culture medium was centrifuged at 4°C and 3000 r / min for 10 min, and the cell supernatant was collected. The contents of NO, IL-6, IL-1β, and TNF-α in the co-culture supernatant were measured using a kit (Xiamen Lunchangshuo Biotechnology Co., Ltd.).

[0121] Results and Discussion

[0122] Table 7 records the effects of the blank control group (only cells), the positive control group (LGG + cells), and the experimental group (strain AUY2301 + cells) on the release of cytokines by macrophages. As shown in Table 7, compared with the LGG treatment group, the intervention of strain AUY2301 significantly increased the levels of IL-6, IL-1β, and TNF-α. Compared with the blank control group, when cells were treated with AUY2301, the release amounts of IL-6, IL-1β, TNF-α, and NO were 37.75 ± 1.56 pg / mL, 37.39 ± 0.73 pg / mL, 205.31 ± 5.51 pg / mL, and 31.89 ± 1.04 μg / mL, respectively. It shows that strain AUY2301 has the effect of activating immune cells and enhancing immunity.

[0123] Table 7

[0124]

[0125]

[0126] Example 7 Evaluation of the anti-inflammatory ability of strain AUY2301

[0127] Effects of AUY2301 on cytokines and prostaglandin E2: Macrophages RAW264.7 were inoculated into 24-well plates, with 1 mL of cell suspension per well (cell concentration was 5×10 5 cells / mL), and cultured at 37°C and 5% CO 2 for 24 h. The cells adhered and grew. The culture medium was discarded. A negative control group (only cells), a model group (cells + LPS), a positive control group (LGG + cells + LPS), and an experimental group (strain AUY2301 + cells + LPS) were set up, with 6 replicate wells in each group. They were cultured at 37°C and 5% CO 2 for 12 h. The culture medium was centrifuged at 4°C and 3000 r / min for 10 min, and the cell supernatant was collected. Kits (Xiamen Lunchangshuo Biotechnology Co., Ltd.) were used to measure the contents of NO, IL-6, IL-1β, TNF-α, and PGE2 in the co-culture supernatant.

[0128] Effects of AUY2301 on the expression of COX-2 in LPS-induced macrophages: Macrophages RAW 264.7 were cultured in 6-well plates at a density of 1.0×10 5 cells / mL, and the selected bacterial suspension (10 9Treated with CFU / mL), with or without lipopolysaccharide (2.5 μg / mL). After 16 hours of incubation, the supernatant was carefully removed, and the cells were lysed in radioimmunoprecipitation assay buffer containing 1% protease phosphatase inhibitor mixture, containing 1% protein phosphatase inhibitor mixture, centrifuged at 8000×g for 20 minutes at 4°C, and the protein concentration in the cell lysate was measured. Equal amounts of protein (15 g) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene fluoride membrane, and then blocked with Tris-buffered saline (10 mmol / L Tris-Cl, pH 7.4) containing 0.5% Tween-20 and 5% skim milk powder for 1 hour at room temperature. The blots were probed with the corresponding primary antibodies (antibodies against COX-2 protein and endogenous GAPDH) overnight at 4°C. After incubation with the primary antibody, the membrane was incubated with a 1:2000 dilution as the secondary antibody. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) detection system.

[0129] Effect of AUY2301 on the release of NO, IL-6, IL-1β and TNF-α in LPS-induced macrophages: Macrophages RAW264.7 were seeded in 24-well plates, with 1 mL of cell suspension per well (cell concentration of 5×10 5 cells / mL), and cultured at 37°C and 5% CO 2 for 24 h. The cells adhered and grew. The culture medium was discarded, and a negative control group (only cells), a positive control group (LGG + cells) and an experimental group (strain AUY2301 + cells) were set up, with 6 replicate wells in each group. The cells were cultured at 37°C and 5% CO 2 for 12 h. The culture medium was centrifuged at 4°C and 3000 r / min for 10 min, and the cell supernatant was collected. Kits (Xiamen Lunchangshuo Biotechnology Co., Ltd.) were used to measure the contents of NO, IL-6, IL-1β and TNF-α in the co-culture supernatant.

[0130] Results and Discussion

[0131] The results are shown in Table 8: The results showed that AUY2301 could significantly inhibit the release of cytokines such as IL-6, IL-1β, TNF-α, NO, PGE 2 . The content of inflammatory factors in the AUY2301 experimental group decreased compared with that in the model group. It indicated that AUY2301 had anti-inflammatory ability.

[0132] Furthermore, in order to study whether the strain AUY2301 participated in the regulation of COX-2 protein related to PGE2 production, Western blot analysis was also used in the present invention to detect their expression. As Figure 1As shown, the expression level of COX-2 protein after LPS stimulation was significantly higher than that of the control group (P<0.05). After pretreatment with AUY2301, the expression of COX-2 protein gradually decreased (P<0.05). These results indicate that AUY2301 inhibits PGE 2 production.

[0133] Table 8

[0134]

[0135] Example 8 Investigation of the Inflammatory Mechanism of AUY2301 by Western Blotting

[0136] The characteristics of the inflammatory response are the coordinated activation of various signaling pathways, regulating the expression of pro-inflammatory and anti-inflammatory mediators in resident tissue cells and leukocytes recruited from the blood. Among them, NF-κB, as a classical inflammatory signaling pathway, has three activation methods, but all rely on kinases. One pathway is triggered by pro-inflammatory cytokines (such as TNF-α). When TNF-α binds to TNF Receptor 1 (TNFR1), the receptor releases Silencer Of Death Domains (SODD) and triggers the sequential recruitment of adapter-like TNF Receptor Associated Death Domain protein (TRADD), Receptor Interacting Protein (RIP), and TNF Receptor Associated Factor (TRAF) to the membrane. TRAF2 recruits the IKK complex to the TNFR1 signaling complex, and then RIP and TRAF2 enter the cytoplasmic membrane. Subsequently, the recruitment and activation of the Inhibitor of nuclear factor kappa-B Kinase (IKK) complex occur. This complex includes the scaffold protein NF-κB essential modulator, IKKα, and IKKβ kinases. Once activated, the IKK complex phosphorylates IκBα (inhibitor of kappa B kinase alpha) at Ser32 and Ser36, followed by ubiquitination and degradation through the proteasome pathway. Finally, the heterodimer p50–p65 is released and migrates to the nucleus, where it binds to specific κB sites and activates multiple NF-κB (nuclear factor kappa-B) target genes (including TNF-α and IL-6). This is the classical activation pathway of NF-κB. We can infer whether NF-κB is activated by detecting changes in the phosphorylation of NF-κB and IκBα proteins, thereby promoting the release of inflammatory factors.

[0137] Culture macrophage RAW 264.7 in a 6-well plate at a density of 1.0×10 5 cells / mL and use the selected bacterial suspension (OD 600nmTreated with 1 μg / mL of LPS (lipopolysaccharide) with or without 2.5 μg / mL of LPS. After incubation for 16 hours, the supernatant was carefully removed, and the cells were lysed in radioimmunoprecipitation assay buffer containing 1% protease phosphatase inhibitor mixture, containing 1% protein phosphatase inhibitor mixture, centrifuged at 8000×g for 20 minutes at 4°C, and the protein concentration in the cell lysate was measured. Equal amounts of protein (15 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene fluoride membrane, and then blocked with Tris-buffered saline (10 mmol / L Tris-Cl, pH 7.4) containing 0.5% Tween-20 and 5% skim milk powder for 1 hour at room temperature. The blot was probed with the corresponding primary antibodies (antibodies against NF-κB and IκBα protein phosphorylation) overnight at 4°C. After incubation with the primary antibody, the membrane was incubated with a 1:2000 dilution as the secondary antibody. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) detection system.

[0138] The results are as Figure 2 shown. The results showed that LPS promoted the phosphorylation of NF-κB and IκBα proteins, while the co-treatment of strain AUY2301 and LPS decreased the phosphorylation levels of NF-κB and IκBα proteins. The results indicated that strain AUY2301 inhibited the release of inflammatory factors through the NF-κB signaling pathway.

[0139] Example 9 Evaluation of the Effect of AUY2301 on the Growth of Caenorhabditis elegans

[0140] (1) Cultivation, passage and synchronization of Caenorhabditis elegans

[0141] Caenorhabditis elegans (wild type N2) and Escherichia coli (E. coli OP50) were provided by Hunan Agricultural University. E. coli OP50 was cultured in (LB) medium and maintained at 37°C. Gravid nematodes were collected in 2 ml centrifuge tubes and washed three times with M9 buffer solution. Then they were transferred to 1 ml of M9 buffer solution for enrichment. The nematodes were treated with a solution composed of 1 M NaOH and 10% NaClO (in a ratio of 1:1) and vortexed for 5 - 8 minutes until the solution became clear. After centrifugation at 3000 g for 3 minutes, the supernatant was discarded. The collected nematode eggs were washed 3 - 4 times with M9 buffer solution. An appropriate amount of nematode eggs was inoculated onto nematode growth medium (NGM) agar plates containing E. coli and incubated at 20°C for 4 days until the nematode eggs reached the adult L4 stage. Subsequently, the synchronized adults were placed on NGM agar plates containing E. coli (OP50) and Lactiplantibacillus plantarum AUY2301 and maintained at 20°C.

[0142] (2) Screening experiment for the concentration of LPS-stimulated oxidative stress model in Caenorhabditis elegans

[0143] To evaluate the effect of LPS (Sigma, Germany) treatment on Caenorhabditis elegans, C. elegans was exposed to different concentrations of LPS at 50, 100, 500, and 1000 μg / mL. The number of dead C. elegans was recorded, and a survival curve for 24 hours was plotted. The absence of movement response upon gentle touch and / or the absence of pharyngeal pumping were considered indicators of C. elegans death. Each treatment was performed 3 times, and approximately 30 C. elegans specimens were used for each treatment.

[0144] Results and Discussion:

[0145] As Figure 3 shown, after treatment with LPS at concentrations of 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL, the survival rate of C. elegans decreased significantly. In addition, the survival rate of C. elegans decreased in a dose- and time-dependent manner. Therefore, we selected 100 μg / mL as the modeling concentration for this experiment.

[0146] (3) Lifespan experiment

[0147] Adult worms (wild-type N2, L4 stage) were exposed to LPS (100 μg / mL) for 24 hours. The adult worms were placed on NGM plates containing OP50 and different concentrations of Lactiplantibacillus plantarum AUY2301 and cultured at 20 °C (starting from day 0). Each treatment was performed with 3 replicates (plates), and at least 30 worms were placed on each plate. At the start of the experiment, 0.3 mL of the bacterial suspension was evenly spread in the center of each plate to attract the worms and prevent them from escaping. Basically, any worms that escaped from the culture dish or showed unnatural death were not included in the count of live worms. During the entire investigation, the number of naturally dead worms in each group was recorded daily, and the surviving worms were transferred to fresh culture dishes. To calculate the average lifespan of worms in each treatment group, the total lifespan of all worms was divided by the number of initial worms in that group at the start of the experiment. Worms that crawled out of the culture dish or died in an unnatural manner were excluded from the calculation.

[0148] Results and Discussion:

[0149] As Figure 4 shown, compared with the LPS model group, the lifespan curves of the Lactiplantibacillus plantarum AUY2301 treatment groups at different concentrations shifted significantly to the right, indicating that strain AUY2301 had a significant lifespan-extending effect on LPS-induced oxidative damage in C. elegans (P < 0.05).

[0150] (4) Reproduction experiment

[0151] Adult worms (wild-type N2, L4 stage) were exposed to LPS (100 μg / mL) for 24 hours. The worms were initially placed on plates containing OP50 and different concentrations of Lactiplantibacillus plantarum AUY2301, and then transferred to fresh NGM plates every 24 hours. After incubation at 20 °C for 48 hours, the plates containing eggs were carefully examined. On day 1, one worm was placed on each plate. On day 2, the fertility of the worms was evaluated. The number of progeny of each worm was counted, and the average number of progeny per group was calculated. The whole process was repeated three times, with at least 10 worms in each group.

[0152] Results and Discussion:

[0153] As Figure 5 shown, compared with the LPS group, the total number of offspring per worm of Caenorhabditis elegans fed different concentrations of AUY2301 increased significantly, indicating that AUY2301 can enhance the reproductive ability of worms under LPS-induced oxidative stress (P<0.05).

[0154] (5) Assessment of locomotor ability

[0155] Locomotion assays were performed using the WormLab automated multi-worm tracking system (MBF Bioscience) at room temperature. Specifically, adult worms (wild-type N2, L4 stage) were exposed to LPS (100 μg / mL) for 24 hours. The worms were initially placed on plates containing OP50 and different concentrations of Lactiplantibacillus plantarum AUY2301, and then moved to fresh plates every 24 h. On days 1, 4, and 7, the worms were transferred to NGM and videotaped for 1 minute. Then, various characteristics of their locomotor behavior were analyzed using WormLab software. The WormLab data were exported to Prism (GraphPad), and the Kruskal-Wallis test was used, followed by Dunn's multiple comparisons to calculate the statistical significance between groups.

[0156] Results and Discussion:

[0157] As Figure 6 shown, the locomotor ability of worms significantly decreased after LPS treatment (P<0.05). Compared with the LPS group, feeding AUY2301 had no significant effect on the locomotor rate of worms in the early and late stages (P<0.05). However, in the middle stage, the locomotor rate increased significantly, showing a dose-dependent relationship (P<0.05).

[0158] (6) Effects of AUY2301 on ROS, SOD, CAT, and MDA in Caenorhabditis elegans

[0159] Wild-type N2 and L4-stage adult worms were respectively exposed to LPS (100 μg / mL) for 24 h. Synchronized adult worms were fed with Lactiplantibacillus plantarum AUY2301 for 10 d, washed three times with M9 buffer, and then H 2 DCF-DA was added to a final concentration of 100 μg / mL in 100 mL. Incubate at 20 °C for 40 minutes, and then use NaN 3 to anesthetize the worms, place them on a glass slide, and observe under an upright fluorescence microscope BX53 (Olympus, Japan). At least 20 worms were analyzed in each group, and the images were analyzed using Image-Pro Plus 6.0 software.

[0160] Adult worms (wild-type N2, L4 stage) were exposed to LPS (100 μg / mL) for 24 h. After feeding the worms with Lactiplantibacillus plantarum AUY2301 for 10 days, the worms were rinsed three times with M9 buffer and then sonicated on ice. The activity levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured according to the instructions of a commercial kit (Nanjing Jiancheng, China).

[0161] Catalase has the ability to rapidly decompose H 2 O 2 . After adding ammonium molybdate, the decomposition by catalase can be rapidly stopped. The remaining H 2 O 2 reacts with ammonium molybdate to form a pale yellow complex. The amount of this complex is measured at a wavelength of 405 nm to determine the CAT activity. The presence of malondialdehyde and thiobarbituric acid (TBA) results in the formation of a red compound, and the absorbance at 532 nm is used to quantify the MDA content.

[0162] Results and Discussion:

[0163] As shown in Table 9, to deeply investigate the effect of AUY2301 on alleviating oxidative damage in Caenorhabditis elegans and reveal the mechanism by which it enables worms to withstand oxidative and heat stress, we used the cell-permeable fluorescent dye H 2 DCF-DA to evaluate the intracellular reactive oxygen species (ROS) level in wild-type worms. Comparative analysis found that the fluorescence intensity in the LPS group increased significantly by 112.65% compared with the OP50 control group. Subsequently, after feeding the worms with different gradient concentrations of AUY2301, the fluorescence intensity in the worms decreased significantly by 71.39%, 80.95%, and 92.15% respectively.

[0164] To explore the potential association between the effects of AUY2301 on LPS-treated Caenorhabditis elegans and oxidative stress, we measured the levels of MDA, CAT, and SOD in the nematodes. When the nematodes were fed with AUY2301 at OD600nm = 0.6, 0.8, and 1.0, the MDA content in the nematodes was 4.56 ± 2.25 nmol / mg, 3.67 ± 0.89 nmol / mg, and 2.98 ± 0.37 nmol / mg, respectively. The CAT content in the nematodes was 186.32 ± 0.87 μg / mL, 199.43 ± 2.09 μg / mL, and 208.98 ± 0.96 μg / mL, respectively. The SOD content in the nematodes was 102.32 ± 0.45 μg / mL, 113.54 ± 0.37 μg / mL, and 123.01 ± 1.28 μg / mL.

[0165] Table 9

[0166]

[0167]

[0168] The above are 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 Lactobacillus plantarum ( Lactiplantibacillus plantarum ) strain AUY2301, characterized in that The plant lactobacillus strain AUY2301 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.30125.

2. A microbial preparation, characterized in that: comprising the plant lactobacillus according to claim 1 ( Lactiplantibacillus plantarum ) strain AUY2301.

3. Use of the plant lactobacillus strain AUY2301 according to claim 1 or the microbial preparation according to claim 2 in the preparation of a medicine having the following effects: Bacteriostatic; and / or Modulate immune activity; and / or Anti-inflammatory; and / or Alleviate oxidative damage; in, The antibacterial function is to inhibit Listeria and Escherichia coli; The regulating immune activity is to promote cells to release immune cytokines; The anti-inflammatory effect is to inhibit the release of inflammatory factors; The mitigation of oxidative damage is: improving the lifespan, reproductive capacity, and mid-life movement capacity of the oxidatively damaged nematodes, and inhibiting the release of reactive oxygen species (ROS) and malondialdehyde (MDA) in the oxidatively damaged nematodes; The promoting cells to release immune cytokines refers to promoting healthy cells to release immune cytokines, and the immune cytokine is at least one of IL-6, IL-1β, TNF-α and NO; The inhibiting the release of inflammatory factors refers to inhibiting the release of inflammatory factors in inflammatory modeling cells, and the inflammatory factors are at least one of IL-1β, TNF-α, NO and PGE2.

4. A fermented product, characterized in that The fermented product is obtained by fermenting the Lactobacillus plantarum strain AUY2301 according to claim 1 or the microbial preparation according to claim 2.

5. A food, characterized in that: The food includes: At least one of the plant lactobacillus strain AUY2301 according to claim 1, the microbial preparation according to claim 2, and the fermented product according to claim 4; and acceptable excipients.

6. A medicine, characterized in that: The drugs include: At least one of the plant lactobacillus strain AUY2301 according to claim 1, the microbial preparation according to claim 2, and the fermented product according to claim 4; and acceptable excipients.

7. A nutritional health product, characterized in that: The nutritional supplements include: At least one of the plant lactobacillus strain AUY2301 according to claim 1, the microbial preparation according to claim 2, and the fermented product according to claim 4; and acceptable excipients.

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