Bifidobacterium bifidum and application thereof

Abstract

The invention discloses a bifidobacterium bifidum LC001 strain and application of the bifidobacterium bifidum LC001 strain in preparation of antioxidant, anti-inflammatory and neuroprotective products. According to the invention, an LC001 strain with potential of antioxidation, anti-inflammation and neuroprotection effects is separated from excrement of healthy human beings, the strain is preserved in China General Microbiological Culture Collection Center on July 8, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.31197. Researches show that the LC001 strain has ORAC oxygen free radical scavenging ability and shows good antioxidant activity; the LC001 strain can be used for remarkably reducing the secretion of inflammatory factors in RAW264.7 and BV-2 cell models induced by lipopolysaccharide (LPS); the LC001 strain is subjected to anaerobic culture, and the content of gamma-aminobutyric acid in fermentation liquor is 5.43 g / L. Therefore, the strain can be used as a probiotic for preparing or developing products with antioxidant, anti-inflammatory and neuroprotective effects.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and more particularly to a bifidobacterium bifidum and application thereof. Background Art

[0002] Inflammation is a coordinated process caused by microbial infection or tissue damage, and is the basis of a variety of physiological and pathological processes. This process involves a large amount of energy metabolism consumption, damage and destruction of host tissues, and may even pose a risk of multiple organ failure and death. The main function of inflammation is to resolve infection or repair damage, restore the body to a state of balance, and create a new environment for tissues. Neuroinflammation is a state caused by autoimmunity, innate immune dysfunction, or infection of the nervous system. Studies have shown that neuroinflammation is closely related to neurodegenerative and neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease, autism, depression, and anxiety.

[0003] Inflammation and oxidative stress play a key role in the development of many diseases. An imbalance in the immune system promotes the production of reactive oxygen species, which further leads to the release of pro-inflammatory cytokines such as interleukin 6 (IL-6), interleukin 1β (IL-1β), and nitric oxide (NO). In some chronic infections or inflammatory diseases, the damage caused by the inflammatory response to the host may be greater than the effect of the microorganisms, which also proves the importance of maintaining the body's homeostasis.

[0004] Long-term imbalance between the production and elimination of free radicals can lead to oxidative stress in the body. Studies have shown that probiotics can improve the body's antioxidant capacity, such as Lactobacillus acidophilus (L.acidophilus), Bifidobacterium longum (S.thermophilus), Bifidobacterium longum (B.longum), Lactobacillus bulgaricus (L.bulgarcicus), etc.; while the body's inhibitory effect on oxygen free radicals is weakened, which will lead to excessive accumulation of oxygen free radicals in the body. Excessive oxygen free radicals can increase the permeability of cell membranes, mobilize the body's inflammatory response, and thus promote the production of pro-inflammatory cytokines in the body. Studies have reported that probiotics can regulate inflammatory responses in a variety of ways. One of the important ways is to regulate the secretion of cytokines to exert anti-inflammatory effects. For example, Lactobacillus rhamnosus LR32, Bifidobacterium lactis BL04, Bifidobacterium longum BL05, Enterococcus faecium Lac.5, etc. can upregulate anti-inflammatory cytokines and downregulate the release of pro-inflammatory cytokines to exert anti-inflammatory effects. Since most strains have a single functional efficacy, it is of great significance to explore probiotics that have antioxidant, anti-inflammatory, and neuroprotective effects for the prevention and treatment of inflammatory and neuroprotective diseases.

[0005] Bifidobacterium is one of the earliest microorganisms to colonize the human intestine and begins to interact with the host during infancy. Some members of the genus Bifidobacterium are believed to play various health-promoting roles at the local and systemic levels. Among them, Bifidobacterium bifidum is the dominant species in the intestinal population of healthy breastfed infants and is also a common member of the intestinal Bifidobacterium population of healthy adults. According to research reports, Bifidobacterium bifidum has functions including antibacterial, regulating lipid metabolism, enhancing the body's immune function, and maintaining the balance of intestinal flora. However, at present, its antioxidant, anti-inflammatory, and neuroprotective effects are not significant. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a Bifidobacterium bifidum and its application, and to provide a Bifidobacterium bifidum LC001 and its application in anti-oxidation, anti-inflammatory and neuroprotection.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: the strain Bifidobacterium bifidum LC001 is deposited in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is July 8, 2024, and the preservation number is CGMCC NO.31197.

[0008] Furthermore, the bacterial 16SrDNA gene sequence is shown in SEQ ID NO.1.

[0009] The invention discloses an application of Bifidobacterium bifidum as a probiotic in the preparation of an antioxidant, anti-inflammatory and neuroprotective product.

[0010] Furthermore, the antioxidant activity refers to the ability to scavenge ORAC oxygen free radicals in vitro; the anti-inflammatory activity refers to reducing the LPS-induced inflammatory response of RAW264.7 cells; and the neuroprotection activity refers to reducing the LPS-induced neuroinflammatory response of BV-2 cells and producing high levels of γ-aminobutyric acid under anaerobic fermentation conditions.

[0011] Furthermore, the LPS is a major component of the cell wall of Gram-negative bacteria (G-), including lipid A, O-specific side chains, and core polysaccharides.

[0012] Furthermore, the inflammation is inflammation in obesity, diabetes, cardiovascular disease, non-alcoholic fatty liver disease, inflammatory bowel disease, and cancer.

[0013] Furthermore, the neuroinflammation includes inflammation in neurodegenerative diseases such as autism, depression, Alzheimer's disease, Parkinson's disease, etc.

[0014] Furthermore, the product is one of functional food, health food, medicine, feed or its additive, and its product form includes one of powder, tablet, granule, capsule and liquid.

[0015] In summary, the present invention has the following beneficial effects: The Bifidobacterium bifidum LC001 strain provided by the present invention has antioxidant, anti-inflammatory and neuroprotective effects. The strain can enhance antioxidant capacity, significantly inhibit the secretion level of inflammatory factors in LPS-induced RAW264.7 and BV-2 cell inflammation models, and produce high levels of γ-aminobutyric acid under anaerobic fermentation conditions. This indicates that LC001 can be used to prepare or develop products with antioxidant, anti-inflammatory and neuroprotective effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the ability curve of each component of the LC001 probiotic of the present invention to scavenge ORAC free radicals; Figure 2 The survival rate of RAW264.7 cells and BV-2 cells in the supernatant of probiotic LC001 at different concentrations; Figure 3 The cell proliferation of RAW264.7 cells and BV-2 cells before and after LPS induction; Figure 4 The figure shows the NO content in the cell supernatant of the RAW264.7 inflammatory model induced by LPS after being treated with the supernatant of probiotic LC001 within a relatively safe concentration range for 24 hours; Figure 5 ELISA determination of IL-6 in the supernatant of RAW264.7 cells after 24 h of treatment with LC001 supernatant in the blank control group (CK), LPS-induced group, and LC001-treated group ( Figure 5 a) and IL-1β ( Figure 5 b) Secretion volume graph; Figure 6 ELISA determination of IL-6 in the cell supernatant of the blank control group (CK), LPS-induced group, and LC001-treated group after 24 hours of treatment of BV-2 cells with LC001 supernatant. Figure 6 a) and IL-1β ( Figure 6 b) Secretion volume graph; Figure 7 This is the liquid chromatogram of LC001 fermentation broth. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0018] The reagents or formulations used in the following examples are: Specific screening medium: weigh 10.0 g / L of hydrolyzed casein, 5.0 g / L of plant peptone, 2.0 g / L of yeast powder, 5.0 g / L of glucose, 0.5 g / L of L-cysteine, 2.0 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium chloride, 0.25 g / L of zinc sulfate, 0.15 g / L of calcium chloride, 0.0001 g / L of ferric chloride, 1.0 g / L of Tween 80, 1 ml / L of glacial acetic acid, 0.05% of L-cysteine, and 50 mg / L of mupirocin and dissolve them in 1000 mL of deionized water, heat and stir until the color becomes yellow and transparent, seal the bottle, and sterilize it by high pressure steam at 121 °C for 20 min. For solid medium, add 15.0 g / L of agar.

[0019] MRS medium: weigh 10.0 g / L peptone, 5.0 g beef extract powder, 4.0 g / L yeast extract powder, 20.0 g / L glucose, 2.0 g / L potassium dihydrogen phosphate, 2.0 g / L ammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, Dissolve 1.0 g / L Tween 80 and 0.05% L-cysteine ​​in 1000 mL of deionized water, heat and stir until yellow and transparent, seal the bottle and sterilize with high pressure steam at 121 °C for 20 min. Add 15.0 g / L agar to the solid culture medium.

[0020] Preparation of 75 mM phosphate buffer: weigh 10.88 g of disodium hydrogen phosphate dodecahydrate and 1.11 g of sodium dihydrogen phosphate dihydrate into a 500 mL volumetric flask, and dissolve them by ultrasonication for later use.

[0021] Preparation of FL fluorescent agent: Accurately weigh 3.2 mg FL fluorescent agent into 200 mL buffer, mix thoroughly and then pipette 100 μL into 20 mL buffer. Prepare before use and keep away from light.

[0022] AAPH preparation: Accurately weigh 0.828 g AAPH and add it to 20 mL buffer and mix well.

[0023] Mouse RAW264.7 cells were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.

[0024] Mouse BV-2 microglia cells were purchased from Wuhan Pronocell Life Science Co., Ltd.

[0025] Nitric oxide (NO) content detection kit: purchased from Bio-Tech Biotechnology Co., Ltd.

[0026] Mouse IL-6 and IL-1β ELISA kits: purchased from Yikesai Biotechnology (Taicang) Co., Ltd.

[0027] Example 1 Isolation and identification of strains Isolation of strains Use sterile oxygen-free saline to gradient dilute the stool sample, and dilute it in sequence according to the gradient dilution method to obtain a diluted bacterial solution with a concentration gradient ranging from 10-1 to 10-6. Take three gradient bacterial solutions of 10-4, 10-5, and 10-6 and apply them on the specific screening culture medium. After applying evenly, turn the plate upside down and place it in an anaerobic culture bag for culture. The strain growth time is about 24h-48h. Select and isolate a single colony suspected of Bifidobacterium bifidum from the colony, and transfer it to a new culture medium by streaking to continue culturing. Pick the flat, slightly gray single colony that has grown, use a 10μL pipette tip to gently scratch the surface of the colony, blow it evenly in 10μL pure water, then take 3μL of water and put it into a PCR tube, add universal primers, mix dye, and DD water, and perform PCR amplification.

[0028] Molecular Biology Identification (1) Pipette 3 μL of bacterial solution and prepare the PCR amplification system and run the program according to the following system (see Table 1 and Table 2) for PCR amplification.

[0029] Table 1: PCR reaction system Table 2: PCR reaction program (2) Prepare 1.5% agarose gel and add 10 μL Ultra GelRed (10000×) nucleic acid dye per 100 mL.

[0030] (3) Add 5 μL of the above PCR reaction solution to each well and use DL2000 marker as a band size control. The band size is about 1500 bp, which can be preliminarily determined to be Bifidobacterium bifidum.

[0031] (4) The samples preliminarily identified as Bifidobacterium bifidum were further amplified by PCR using universal primers. The amplified PCR stock solution was sent to Qingke Biotechnology Co., Ltd. for sequencing. After the sequencing results were obtained, the sequences were compared on the website https: / / blast.ncbi.nlm.nih.gov / Blast.cgi to obtain the comparison results.

[0032] (5) After comparison, the samples identified as Bifidobacterium bifidum were expanded and cultured in MRS liquid medium for 48 hours. 100 μL of bacterial solution was aspirated, the bacterial solution was centrifuged and the supernatant was discarded. 100 μL of sterile water was added for re-dissolution, and PCR amplification was performed again. After amplification, it was sent to Qingke Biotechnology for sequencing. If it was identified as a single peak Bifidobacterium bifidum, it was pure Bifidobacterium bifidum. The bacterial solution was mixed with 60% glycerol solution in a 1:1 ratio and stored in a -80°C refrigerator.

[0033] Example 2 Scavenging effect of each component of LC001 on ORAC free radicals 1. Test methods The antioxidant capacity index (ORAC) is a commonly used method for determining antioxidant capacity. In this paper, the in vitro antioxidant capacity of the supernatant, live bacteria, pasteurized, and lysed LC001 strain was determined by this method. The specific method is: 25 μL of sample was aspirated into a 96-well plate, 150 μL of FL fluorescent agent was added, and the mixture was incubated at 37°C in the dark for 15 min, followed by the addition of 25 μL of AAPH, and phosphate buffer was used as a control. The fluorescence intensity was measured and recorded at an excitation wavelength of 485 nm and an emission wavelength of 525 nm, and the reading was taken once every 10 min for a total of 2 h until the fluorescence decayed to the baseline. The larger the ORAC value, the stronger the oxygen free radical scavenging ability of the LC001 strain and the higher its antioxidant activity. Note: In this experiment, the supernatant was diluted 1000 times, and the live bacteria, pasteurized, and lysed were diluted 100 times.

[0034] 2. Test results Figure 1 a is the fluorescence kinetic curve of each component of LC001 probiotics, Figure 1b is the ORAC value of each component of LC001 probiotics. It can be seen that among the four components of supernatant, live bacteria, pasteurized, and lysed, the ORAC value of the supernatant group is the largest, reaching 397.6 mMTrolox, the ORAC value of the live bacteria group is 20.9 mM Trolox, the ORAC value of the pasteurized group is 27.1 mM Trolox, and the ORAC value of the lysed group is 21.9 mM Trolox. This shows that the antioxidant activity of the supernatant of LC001 probiotics is the strongest, followed by pasteurized, lysed, and live bacteria.

[0035] Example 3 Effects of different concentrations of LC001 supernatant on the survival rate of RAW264.7 and BV-2 cells 1. Cell recovery Prepare a 15 mL centrifuge tube and add 9 mL of DMEM basal medium to it in the clean bench. First, preheat the water bath to 37 °C in advance. After taking out the cell cryopreservation tube from the liquid nitrogen tank, immediately put it into the water bath and shake it quickly to melt it in a short time. Use a pipette to slowly transfer the cell suspension to the centrifuge tube in the clean bench and centrifuge it at 1000 rpm for 5 min. At the same time, add 7 mL of DMEM complete medium prepared with 1% triple antibody, 10% fetal bovine serum, and DMEM basal medium to the cell culture dish. After centrifugation, remove the centrifuge tube, discard its supernatant in the clean bench, add 1 mL of DMEM complete medium, resuspend the cells, and then transfer the cells to the culture bottle, gently blow and aspirate, cover the culture dish lid and place it in a 37 °C, 5% CO2 incubator for culture.

[0036] 2. Cell passaging After the cells are revived and placed in the incubator for 24 hours, take out the cells and observe them under a microscope. When the cell growth density is about 80% of the area at the bottom of the culture bottle, the cells can be passaged. When passaged, first pour out the old culture medium in the culture dish, then slowly rinse with PBS buffer solution 2-3 times, discard the PBS solution, add 1mL of trypsin digestion solution, let it stand for 2-3 minutes, and finally add 4mL of DMEM complete culture medium to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, then add 1mL of DMEM complete culture medium, resuspend the cells, blow and mix again, tighten the bottle cap and observe under a microscope, blow the cells gently, cover the culture dish lid and place it in a 37°C, 5% CO2 incubator for culture. If the cells are found to grow slowly and the degree of fusion is not high, the medium can be changed.

[0037] 3. Cell cryopreservation Observe the cell status during culture, and cells in the logarithmic growth phase and in good condition can be frozen. When freezing, carefully aspirate the cell suspension into a centrifuge tube with a pipette, tighten the centrifuge tube cap and centrifuge at 1000 rpm for 5 min, discard the cell supernatant, add 1mL cell freezing solution (DMSO: fetal bovine serum = 1:9), gently pipette the cells to mix, transfer to a cryopreservation tube, mark it, place it in a gradient cooling cryopreservation box, freeze it in a -80℃ refrigerator overnight, and transfer it to a liquid nitrogen tank the next day for long-term freezing.

[0038] CCK-8 cytotoxicity assay (1) Cell plating: RAW264.7 and BV-2 cells with good growth status were plated at 5×10 4 / mL, 100 μL per well was inoculated into a 96-well cell culture plate, and the culture plate was placed in an incubator and cultured until the adhesion rate reached 80%-90%, and then cultured overnight; (2) Co-culture of probiotics and cells: Complete culture medium was used to act on RAW264.7 cells with different concentrations of LC001 supernatant (25%, 12.5%, 6.25%, 3.12%, 1.56%); different concentrations of LC001 supernatant (10%, 5%, 3.3%, 2.5%) were used to act on BV-2 cells; 4 replicate wells were set up for each group and placed in an incubator for 24 h; (3) Add CCK-8: Dilute the CCK-8 solution to 10% with complete culture medium, discard the old culture medium, add 100 µL of 10% CCK-8 solution to each well, place the well in an incubator and culture for 40 min. Measure the absorbance at 450 nm using a microplate reader.

[0039] 5. Test results The results are as follows Figure 2 As shown in a, the survival rate of RAW264.7 cells decreased significantly with the decrease of the concentration of LC001 strain supernatant, showing a concentration-dependent effect. When the concentration of the strain supernatant was 25% and 12.5%, the cell survival rate was low and the cytotoxicity was relatively large. The concentration of 6.25% had a relatively low cytotoxicity. In the future, a safe concentration of 6.25% strain supernatant will be selected to act on RAW264.7 cells; the survival rate results of BV-2 cells are shown in Figure 2 As shown in b, when the concentration of the strain supernatant is 2.5%, the cell survival rate is high and the cytotoxicity is low. Subsequently, a safe concentration of 2.5% of the strain supernatant will be selected to act on BV-2 cells.

[0040] Example 4 Cell proliferation before and after LPS induction 1. Test methods Lipopolysaccharide (LPS) is an important component of Gram-negative bacterial endotoxins and is often used to trigger cell inflammation. Its mechanism of action is through the action of multiple proteins including TLR4. Therefore, the present invention uses LPS as an inducer to establish an LPS-induced RAW264.7 and BV-2 cell inflammation model. Cells in good growth state were taken at 5×10 4 / mL, 100μL per well was inoculated in a 96-well cell culture plate. When the cell adhesion rate reached 80%-90%, the culture medium was discarded and the LC001 strain was treated. A blank control group and an LPS-induced group were set up, with 6 replicates in each group. 200μL of complete culture medium was added to each well of the blank control group; 200μL of complete culture medium containing 2 μg / mL LPS was added to each well of the LPS-induced group. After 24h, the survival rate of RAW264.7 and BV-2 cells was detected by CCK-8 method.

[0041] Test results The results are as follows Figure 3 As shown in a, after RAW264.7 cells were co-cultured with LPS, the number of cells increased compared with the blank control group, and there was no cytotoxicity; the survival rate of BV-2 cells was as shown in Figure 3 As shown in b, the number of cells in the LPS group increased compared with the blank group, and there was no cytotoxicity. This indicates that LPS at low concentrations has a low cell survival rate for RAW264.7 and BV-2 cells. Taking all factors into consideration, 2 µg / mL of LPS was subsequently used to act on RAW264.7 and BV-2 cells.

[0042] Example 5 Effect of LC001 supernatant on NO content in LPS-induced RAW264.7 cells 1. Test methods After the safe concentration range of LC001 supernatant was determined according to Example 3, the safe concentration of LC001 supernatant was co-cultured with 2 μg / mL LPS. RAW264.7 cells with good growth status were taken and cultured at 5×10 4 / mL, 100μL per well was inoculated in a 96-well cell culture plate. When the cell adhesion rate reached 80%-90%, the culture medium was discarded and the treatment was carried out. A blank control group, an LPS-induced group, and an LC001 supernatant-treated group were set up, with 6 replicate wells in each group. In the blank control group, 200μL of complete culture medium was added to each well; in the LPS-induced group, 200μL of complete culture medium containing 2μg / mL LPS was added to each well; in the LC001 supernatant group, 200μL of complete culture medium containing 6.25% LC001 supernatant and 2μg / mL LPS was added to each well. After 24h, the cell supernatant was collected, and the NO content in the supernatant of RAW264.7 cells was determined using a nitric oxide detection kit. The detailed steps are as follows: The 1M NaNO2 standard was serially diluted to 100, 60, 40, 20, 10, 5, 2, and 1 μM using 1640 basal medium containing 10% fetal bovine serum.

[0043] The collected cell culture supernatant was centrifuged at 3000 rpm for 5 min to remove the cell precipitate; Three parallel samples were added to each well of a 96-well plate at 50 μL / well. 50 μL of Griess reagent I was added to each well. After reacting for 5-10 minutes, 50 μL of Griess reagent II was added. The plate was tapped to mix well and reacted for 5 minutes. The OD value was measured at 540 nm on an enzyme-labeled instrument, and the corresponding content in the sample could be calculated after establishing a NO standard curve.

[0044] 2. Test results The results are as follows Figure 4 As shown, the secretion of NO in the LPS-induced group was significantly increased compared with the CK group (p < 0.01). After treatment with the supernatant of the LC001 strain, the secretion of NO in the LPS-induced RAW264.7 cells was significantly reduced (p < 0.01), down by 77.7%.

[0045] Example 6 Effect of LC001 supernatant on IL-6 and IL-1β levels in LPS-induced RAW264.7 cells 1. Test methods RAW264.7 cells in good growth condition were used at 2×10 6 / mL, 2mL per well was inoculated on a six-well cell culture plate. After the cells were firmly attached to the wall, the supernatant was discarded and a blank control group (CK), LPS-induced group, and LC001 supernatant treatment group were set up. 2mL of complete culture medium was added to each well of the blank control group; 2mL of complete culture medium containing 2μg / mL LPS was added to each well of the LPS-induced group; and 2mL of complete culture medium containing 6.25% LC001 supernatant and 2μg / mL LPS was added to each well of the LC001 supernatant group. After 24h, the cell supernatant was collected, and the secretion of IL-6 and IL-1β in the cell supernatant was determined using a mouse IL-6 and IL-1β ELISA kit. Please refer to the kit instructions for the detection steps. The detailed steps are as follows: Take the kit out of the refrigerator in advance and return it to room temperature. Dilute the concentrated washing solution with ultrapure water at a ratio of 1:20, and dilute the biochemical antibody working solution and enzyme conjugate solution with diluent at a ratio of 1:100. After the enzyme conjugate solution is prepared, place it in a light-proof room 30 minutes before use. The concentrations of IL-6 and IL-1β standards in the gradient dilution kit are: 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.625pg / mL, 0pg / mL. Determine the concentrations of IL-6 and IL-1β in the sample through preliminary experiments and dilute them to the appropriate standard curve concentration range for subsequent determination; The collected cell supernatant was centrifuged at 3000 rpm for 5 min. After centrifugation, gradient dilution was performed according to the preliminary experimental results. Set up blank wells (zero wells), standard wells and sample wells (CK, LPS, LC001 supernatant) on the experimental strips, and set up three parallels for each group. Add different concentrations of standard products and samples to the corresponding wells, 100μL per well, and then add 50μL of biochemical antibody working solution to each well, without adding to the zero wells, and seal the reaction wells with sealing tape, and incubate at room temperature at 300rpm for 120min; Shake off the liquid in the wells, pat dry the remaining liquid on a thick stack of clean absorbent paper, add 350 μL of washing solution to each well, let stand for 30 seconds, shake off the liquid in the wells, and repeat the washing 5 times; Add 100 μL of enzyme conjugate solution, do not add to the zero well, seal the reaction well with sealing tape, and incubate at room temperature at 300 rpm for 120 min; Repeat step (5). Including the zeroed well, 100 μL of chromogenic substrate was added to each well and incubated at room temperature in the dark for 15 min; Add 100 μL of reaction stop solution to each well, including the zero well, and measure the OD450nm value within 10 minutes after mixing the system.

[0046] 2. Test results The results are as follows Figure 5 As shown in the figure, compared with the blank control group (CK), LPS (2 μg / mL) induced RAW264.7 cells, significantly increased the levels of inflammatory factors IL-6 and IL-1β in the cell supernatant (p < 0.01). After treatment with LC001 supernatant, the LPS-induced inflammatory effect was significantly weakened. For the inflammatory factor IL-6, as Figure 5 As shown in a, LC001 supernatant treatment significantly attenuated the inflammatory level induced by LPS by 90.0%; for IL-1β, Figure 5 As shown in b, LC001 supernatant treatment significantly reduced the inflammation by 35.5%, indicating that it has effective therapeutic ability against LPS-induced RAW264.7 cell inflammation.

[0047] Example 7 Effect of LC001 supernatant on IL-6 and IL-1β levels in LPS-induced BV-2 cells 1. Test methods BV-2 cells in good growth condition were used to generate 2×10 6 / mL, 2mL per well was inoculated on a six-well cell culture plate. After the cells were firmly attached to the wall, the supernatant was discarded and a blank control group (CK), LPS-induced group, and LC001 supernatant treatment group were set. The blank control group added 2mL of complete culture medium to each well; the LPS-induced group added 2mL of complete culture medium containing 2μg / mL LPS to each well; the LC001 supernatant group added 2mL of complete culture medium containing 2.5% LC001 supernatant and 2μg / mL LPS to each well. After 24h, the cell supernatant was collected, and the secretion of IL-6 and IL-1β in the cell supernatant was determined using a mouse IL-6 and IL-1β ELISA kit. The detection steps are the same as the detailed steps of the kit in Example 6.

[0048] 2. Test results The results are as follows Figure 6 As shown in the figure, compared with the blank control group (CK), LPS (2 μg / mL) induced BV-2 cells, significantly increased the levels of inflammatory factors IL-6 and IL-1β in the cell supernatant (p < 0.01). Figure 6 As shown in a, LC001 supernatant treatment significantly attenuated IL-1β by 41.8%. Figure 6 As shown in b, LC001 supernatant treatment significantly attenuated the inflammation by 31.6%, indicating that it has effective therapeutic ability against LPS-induced neuroinflammation in BV-2 cells.

[0049] Example 8 Detection of γ-aminobutyric acid production by LC001 under anaerobic conditions 1. Test methods The LC001 glycerol culture stored at -80℃ was inoculated into MRS liquid medium containing 0.05% L-cysteine ​​hydrochloride at a 1% v / v inoculation rate. After culturing at 37℃ for 24h, it was transferred to the fermentation medium at a 5% v / v inoculation rate and cultured at 37℃ under anaerobic conditions. Fermentation medium (L): 10g peptone, 5g beef powder, 4g yeast powder, 2g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 0.5g L-cysteine ​​hydrochloride, 10g L-glutamate sodium. Take the bacterial solution at 48h of fermentation, centrifuge at 12000rpm for 5min, and retain the supernatant. High performance liquid chromatography was used to detect the content of γ-aminobutyric acid in the fermentation broth. The sample to be tested needed to be derivatized before injection. The specific operation was as follows: 100 μL of the sample to be tested was fully mixed with 100 μL of o-phthalaldehyde solution (10 mg o-phthalaldehyde was dissolved in 2.5 mL of acetonitrile and then 20 μL of β-mercaptoethanol was added) and 500 μL of 0.4 M boric acid buffer (0.4 M boric acid, sodium hydroxide adjusted to pH = 9.5), and then allowed to stand for 2 minutes. After filtering through a 0.22 μm filter membrane, liquid phase detection was performed. The liquid phase detection conditions are as follows: Chromatographic column: Hypersil ODS C18 (4.6mm×250mm, 5μm); mobile phase: acetonitrile: 0.02M sodium acetate = 23:77; flow rate: 0.9ml / min; column temperature: 30℃; injection volume: 20μL; detection wavelength: 333nm.

[0050] 2. Test results The liquid chromatogram of LC001 fermentation broth is as follows Figure 7 As shown, after calculation, the content of γ-aminobutyric acid in the fermentation broth of Bifidobacterium bifidum LC001 is 5.43 g / L.

[0051] In summary, the LC001 strain provided by the present invention can enhance the antioxidant capacity, significantly reduce the secretion of inflammatory factors in LPS-induced RAW264.7 and BV-2 cells, produce γ-aminobutyric acid under anaerobic fermentation conditions, and has antioxidant, anti-inflammatory and neuroprotective effects.

[0052] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Bifidobacterium bifidum LC001, which is deposited in the China General Microbiological Culture Collection Center (CGMCC) on July 8, 2024, with a deposit number of CGMCC NO.31197.

2. A strain of Bifidobacterium bifidum according to claim 1, characterized in that: The bacterial 16SrDNA gene sequence is shown in SEQ ID NO.

1.

3. Use of the Bifidobacterium bifidum according to claim 1 as a probiotic in the preparation of antioxidant, anti-inflammatory and neuroprotective products.

4. The use according to claim 3, characterized in that: The antioxidant activity refers to the ability to scavenge ORAC oxygen free radicals in vitro; the anti-inflammatory activity refers to the ability to reduce LPS-induced inflammatory response in RAW264.7 cells; the neuroprotective activity refers to the ability to reduce LPS-induced neuroinflammatory response in BV-2 cells and to produce high levels of γ-aminobutyric acid under anaerobic fermentation conditions.

5. The use according to claim 4, characterized in that: The LPS is a major component of the cell wall of Gram-negative bacteria (G-), including lipid A, O-specific side chains, and core polysaccharides.

6. The use according to claim 4, characterized in that: The inflammation is inflammation in obesity, diabetes, cardiovascular disease, non-alcoholic fatty liver disease, inflammatory bowel disease, and cancer.

7. The use according to claim 4, characterized in that: The neuroinflammation includes inflammation in neurodegenerative diseases such as autism, depression, Alzheimer's disease, Parkinson's disease, etc.

8. The use according to claim 3, characterized in that: The product is one of functional food, health food, medicine, feed or its additive, and its product form includes one of powder, tablet, granule, capsule and liquid.

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