Celine animal bifidobacterium, metagen, pharmaceutical composition and application of feline animal bifidobacterium, metagen and pharmaceutical composition

Abstract

The invention provides a feline animal bifidobacterium, a metagen, a pharmaceutical composition and application, the feline animal bifidobacterium is named as animal bifidobacterium GYJC-001, the strain is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number is CCTCC NO: M 20241621. The invention further provides a preparation method of the feline animal bifidobacterium, the metagen, the pharmaceutical composition and the application of the feline animal bifidobacterium, the metagen, the pharmaceutical composition and the application of the feline animal bifidobacterium, the metagen, the pharmaceutical composition and the application of the feline animal bifidobacterium. The bifidobacterium animalis GYJC-001 disclosed by the invention has the advantages that the acid resistance and cholate resistance are high, the intestinal colonization ability is strong, the probiotic effect is better, the toxic and side effects are avoided, and a certain theoretical basis is provided for the development of the bifidobacterium animalis GYJC-001.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a cat-derived animal bifidobacterium, a postbiotic, a pharmaceutical composition and an application thereof. Background Art

[0002] Domestic pet cats are kept in a stable environment for a long time. Once they are stimulated by changes in the environment and other external factors, they are very likely to have a stress response, which can seriously cause the death of the animal. Improving animal immunity is closely related to resisting pathogenic microorganisms, reducing intestinal diseases, improving metabolism, fur health and wound recovery. As the second largest immune organ in the body, the intestine plays an important role in immune regulation. Therefore, the body's immunity can be improved by improving the health of the intestinal environment.

[0003] Probiotics can beneficially affect the health of host animals by improving the intestinal microecological balance of host animals. Among them, lactic acid bacteria are one of the most important feed probiotics. They use carbohydrates to ferment under facultative anaerobic conditions to produce metabolites such as lactic acid, which can beneficially affect the health of host animals by improving the intestinal microecological balance of host animals. Postbiotics are "preparations of inanimate microorganisms and / or their components that are beneficial to the host", including metabolites and inactivated bacteria of probiotics. Studies have shown that they play a similar or even better role than live probiotics, and because of their clear chemical structure, long shelf life, higher safety and greater stability, they also avoid the risk of live bacteria transferring drug-resistant genes. Bifidobacterium animalis has been widely used as a safe and effective probiotic. A large number of studies have shown that the mechanisms by which Bifidobacterium animalis improves intestinal diseases include inhibiting inflammation and immune responses, regulating intestinal microbiota and its metabolism, and inhibiting oxidative stress. Among them, the basic functions, beneficial effects and potential mechanisms of animal Bifidobacterium in intestinal diseases have been widely concerned and understood, including inhibiting the reproduction of gastrointestinal pathogens, reducing animal stress, regulating intestinal flora, preventing or alleviating diarrhea, and also playing an important role in regulating animal immune function and promoting animal growth.

[0004] The composition of intestinal microorganisms of different species of animals varies greatly, and homologous probiotics often have better colonization ability and effect in the host. At present, the main source of animal bifidobacteria is infant feces, etc., and the probiotics widely used in pet cat breeding are mainly non-cat-derived probiotics. The present invention discloses a preparation and use of cat-derived animal bifidobacteria and postbiotics with anti-inflammatory and antioxidant effects, which are used to prepare animal food compositions or supplement compositions. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a cat-derived bifidobacterium, postbiotics, pharmaceutical composition and application, which are used to solve the problems of the lack of strains of existing feline-derived probiotics, huge differences in application effects, and being unfavorable for controlling animal infections and increasing animal production.

[0006] To achieve the above-mentioned purpose and other related purposes, the present invention provides a cat-derived animal bifidobacterium, named as Bifidobacterium animalis GYJC-001, which was deposited in the China Center for Type Culture Collection (abbreviated as CCTCC, address: Wuhan University, Wuhan, China) on July 22, 2024, with a deposit number of CCTCC NO: M20241621.

[0007] The animal bifidobacterium GYJC-001 of the present invention has the following morphological characteristics: the colony is white or milky white in color, round in shape, with neat edges and a moist and smooth surface.

[0008] Preferably, the 16SrRNA gene sequence of Bifidobacterium animalis GYJC-001 is as shown in SEQ ID NO.1.

[0009] Preferably, the survival rate of the Bifidobacterium animalis GYJC-001 after being treated with a culture solution having a pH of 2.0 to 3.0 for 3 hours is 90% or even above 96%; and the self-aggregation rate after being treated for 12 hours is 50% or even above 55%.

[0010] Preferably, the Bifidobacterium animalis GYJC-001 is isolated from lactic acid bacteria in feces of Chinese garden cats.

[0011] Preferably, the Bifidobacterium animalis GYJC-001 has acid resistance, alkali resistance, self-aggregation ability, and intestinal colonization ability.

[0012] Preferably, the Bifidobacterium animalis GYJC-001 has the ability to inhibit the growth of pathogenic microorganisms, and the pathogenic microorganisms include but are not limited to cat-derived Salmonella Dublin, cat-derived Salmonella Typhimurium, and cat-derived Escherichia coli.

[0013] Preferably, the Bifidobacterium animalis GYJC-001 has the ability to inhibit the production of inflammatory factors by cat kidney cells induced by LPS in vitro and improve the antioxidant capacity of cat kidney cells.

[0014] The present invention also provides an animal bifidobacterium postbiotic, which is a starter, culture, bacterial suspension or lysate of cat-derived animal bifidobacterium.

[0015] The present invention also provides a composition comprising one or more of the above-mentioned cat-derived animal bifidobacterium, its culture, its bacterial suspension and its lysate.

[0016] Preferably, the cat-derived bifidobacterium is a live bacterium or a non-live bacterium, in particular a live bacterium.

[0017] Preferably, the composition further comprises other probiotics and / or prebiotics, for example, other microorganisms and / or prebiotics that are beneficial for regulating the homeostasis of intestinal flora, preventing and treating the occurrence of intestinal diseases such as inflammatory bowel disease.

[0018] Preferably, the composition is a pharmaceutical composition, a food composition, a dietary supplement composition, a feed additive composition or a feed composition.

[0019] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0020] More preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents, fragrances, colorants, coating agents, acidity regulators, preservatives, diluents, suspension stabilizers, and the like.

[0021] Preferably, the pharmaceutical composition can be in any suitable dosage form, in particular an oral dosage form, for example, a liquid preparation (such as a suspension, emulsion, syrup, etc.), a solid preparation (such as a powder, tablet, pill, granule, capsule, lozenge, etc.), etc.

[0022] More preferably, the pharmaceutical composition is a liquid preparation, wherein the animal Bifidobacterium GYJC-001 is a live bacterium, and its effective live bacterial count is ≥ 1×10 9 CFU / mL, for example, 1×10 9 Up to 10×10 9 CFU / mL, 5×10 9 CFU / mL.

[0023] Specifically, the above-mentioned food composition is a food for daily intake. It can be a food composition directly added with animal Bifidobacterium GYJC-001, its culture, its bacterial suspension or its lysate (especially live bacteria of animal Bifidobacterium GYJC-001), or it can be a food composition produced by fermentation after adding animal Bifidobacterium GYJC-001.

[0024] Specifically, the above-mentioned feed additive composition is a substance added in small amounts or trace amounts during the production, processing and use of feed (such as livestock feed, livestock drinking water, pet daily diet, pet snacks, pet drinking water, etc.), which comprises animal Bifidobacterium GYJC-001, its culture, its bacterial suspension or its lysis product (especially animal Bifidobacterium GYJC-001), and one or more additive excipients.

[0025] Specifically, the additive excipient may be, for example, but not limited to, one or more of a filler, a binder, a wetting agent, a lubricant, a flavoring agent, a disintegrant, a colorant, a coating agent, an acidity regulator, a diluent, a suspension stabilizer, a preservative, and the like.

[0026] Specifically, the feed additive composition can be in any form, such as tablets, pills, capsules, powders, granules, liquids, etc.

[0027] Specifically, the above-mentioned feed composition is feed for daily feeding of animals (such as pets, livestock) by humans, for example, livestock feed, pet diet, pet snacks, etc. It can be a feed composition directly added with animal Bifidobacterium GYJC-001, its culture, its bacterial suspension or its lysate (especially live bacteria of animal Bifidobacterium GYJC-001), or it can be a feed composition produced by fermentation after adding animal Bifidobacterium GYJC-001.

[0028] The present invention also provides a use of cat-derived bifidobacteria in the preparation of a drug for preventing and / or treating intestinal diseases. The therapeutic effect may be to improve one or more of the symptoms of the intestinal diseases, reduce the recurrence rate of the intestinal diseases, or even completely cure the intestinal diseases. The preventive effect may be to reduce the incidence of intestinal diseases, or even completely avoid the intestinal diseases.

[0029] As described above, the present invention has the following beneficial effects:

[0030] (1) The present invention provides a Bifidobacterium animalis GYJC-001 derived from Chinese rural cat feces, which not only enriches the lactic acid bacteria resources, but also promotes the development and utilization of cat-derived microbial resources;

[0031] (2) The animal bifidobacterium GYJC-001 of the present invention has the characteristics of high acid resistance and bile salt resistance, strong intestinal colonization ability, good prebiotic effect and no toxic side effects, which provides a certain theoretical basis for the development of animal bifidobacterium GYJC-001;

[0032] (3) The Bifidobacterium animalis GYJC-001 of the present invention can significantly alleviate the inflammation level and oxidative reaction of renal epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the colony morphology of Bifidobacterium animalis GYJC-001 screened out from Chinese garden cat feces.

[0034] Figure 2 The inhibitory effect of BAGYJC-001 on Salmonella Dublin from cats was shown in the following groups: PBS, MRS, CFS (culture supernatant of BA), and GM (gentamicin).

[0035] Figure 3 The inhibitory effect of BAGYJC-001 on Salmonella Typhimurium from cats was shown in the figure. The groups were PBS, MRS, CFS (culture supernatant of BA), and GM (gentamicin).

[0036] Figure 4 The inhibitory effect of BAGYJC-001 on Escherichia coli of cat origin was shown in the figure. The groups were PBS, MRS, CFS (culture supernatant of BA), and GM (gentamicin).

[0037] Figure 5 The effect of BAGYJC-001 on the antioxidant effect of CRFK cells.

[0038] Figure 6 This is the effect of BAGYJC-001 on the production of inflammatory cytokines in CRFK cells induced by LPS. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0041] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0042] The present application embodiment provides a method for obtaining Bifidobacterium animalis GYJC-001, comprising the following steps:

[0043] (1) Preparation of dilution solution:

[0044] Take 1g of fresh Chinese cat feces and dilute it to 10 with sterile PBS. 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 There are seven concentration gradients in total, and three parallel tests are performed for each gradient. After the samples in the dilution solutions of different concentration gradients are completely mixed, the next step of the experiment is carried out.

[0045] (2) Cultivation:

[0046] Take 100 μL of each dilution in the order of dilution concentration from low to high, and inoculate into MRS solid medium (peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, ammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.2g / L, Tween 80 1.0mL / L, glucose 20g / L, agar 20g / L, and the rest is double distilled water; pH value is adjusted to 6.2-6.6 with glacial acetic acid, sterilized at 121℃ for 20 minutes) plates, and evenly spread with a sterile spreader. After absorption, place in a 37℃ constant temperature incubator for anaerobic culture for 48h.

[0047] (3) Isolation, purification and identification of strains

[0048] (S1) Isolation and purification: Take the above plates with various bacteria, select single colonies of lactic acid bacteria, and use the continuous streaking method to separate and purify. Purify single colonies with different colony morphologies. Repeat 3 to 4 times and examine under a microscope until a pure strain is obtained.

[0049] (S2) Observe the characteristics of the colonies and observe the morphology of bacteria using Gram staining and microscopy;

[0050] (S3) DNA extraction: Bacterial DNA was extracted according to the instructions of the Takara DNA extraction kit. The conserved sequences of lactic acid bacteria were amplified and sequenced using the universal primers for bacteria: 27f, 1492r. The primer synthesis and sequencing were completed by Hangzhou Qingke Biotechnology Co., Ltd., and the 16S rRNA sequences were compared for homology.

[0051] (S4) Isolation, screening and identification of animal Bifidobacterium: After morphological observation, microscopic examination and 16S rRNA sequencing, it was identified as animal Bifidobacterium and named GYJC-001. After 48 hours of culture, the colony had a convex middle, neat edges, smooth surface and milky white color; it was facultative anaerobic, Gram-positive, and the cells were bifurcated rod-shaped. The colony morphology of GYJC-001 is as follows Figure 1 shown.

[0052] The 16SrRNA gene sequence of Bifidobacterium animalis GYJC-001 is shown in SEQ ID NO.1.

[0053] (4) Strain preservation:

[0054] The strain deposit information is as follows:

[0055] Deposit name: Bifidobacterium animalis GYJC-001, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC M20241621, deposit date July 22, 2024.

[0056] The present application example also conducts the following research on the strain characteristics of Bifidobacterium animalis GYJC-001:

[0057] After BAGYJC-001 colonies were activated, they were cultured in 50 mL of MRS liquid culture medium (same formula as above) with an initial inoculation concentration of 1.0 × 10 8 CFU / mL, and each bacterial solution was adjusted to 1.0×1010 CFU / mL, and then subcultured in MRS culture medium for 2 to 3 times to obtain the culture medium of the bacteria to be tested.

[0058] [Strain acid resistance]

[0059] Take 0.5 mL of bacterial solution and transfer it to 4.5 mL of simulated gastric fluid with pH = 2.5, and culture it at 39 ° C for 0 h and 3 h. Use the dilution plate coating method to determine the total number of viable bacteria (CFU / mL) in each time period, and use the 0 h measurement value as the blank control to calculate the survival rate. The calculation formula is as follows:

[0060]

[0061] The results are shown in Table 1. The viability of BAGYJC-001 in the medium with pH = 2.5 for 3 hours was above 95%, and it had good acid resistance.

[0062] Table 1. BAGYJC-001 artificial gastric juice tolerance results

[0063] Detection indicators Viable bacteria count (LogCFU / mL) Survival rate (%) 0h 8.69±0.27 3h 8.52±0.04 96.78±1.67

[0064]

Alkali resistance of strains

[0065] Take 0.5mL of bacterial solution and transfer it to 4.5mL of simulated intestinal fluid with pH=8.0, and culture it at 37℃ for 0h and 3h. Use the dilution plate coating method to determine the total number of viable bacteria (CFU / mL) in each time period, and use the 0h measurement value as the blank control to calculate the survival rate. The calculation formula is as follows:

[0066]

[0067] The results are shown in Table 2. The viability of BA GYJC-001 in the medium with pH = 8.0 for 3 hours was above 65%, and it had good alkali resistance.

[0068] Table 2 Results of tolerance of Lactobacillus reuteri to artificial intestinal fluid

[0069] Detection indicators Viable bacteria count (logCFU / mL) Survival rate (%) 0h 9.40±0.06 3h 6.18±0.21 65.74±2.24

[0070]

High temperature resistance of strains

[0071] Take 200μL of the activated bacterial solution to be tested and re-inoculate it into 10mL of clean MRS medium, and incubate it at 37℃ overnight. Then, inoculate the activated bacterial solution into 1.5mL sterile centrifuge tubes at 1mL / tube, and place the 1.5mL centrifuge tubes in 37℃, 55℃, and 70℃ water baths respectively, incubate for 5min, and finally use the plate dilution coating method to count the number of live bacteria after 5min of treatment at different temperatures. The calculation formula is as follows:

[0072]

[0073] The results are shown in Table 3. The survival rate of BA GYJC-001 can reach 95% at 55°C and about 69% at 70°C, which shows good high temperature tolerance.

[0074] Table 3 Results of high temperature tolerance of Lactobacillus reuteri

[0075] 37℃5min 55℃5min 70℃5min Viable bacteria count (logCFU / mL) 10.00±0.20 9.51±0.05 6.92±0.31 Survival rate (%) 100 95.20±1.74 69.19±3.21

[0076]

Strain hydrophobicity

[0077] After the strain is activated by liquid culture medium, centrifuge at 8000g for 5 minutes, collect the lower bacterial sediment, wash with sterile PBS three times, resuspend and adjust the optical density of the bacterial suspension at 600nm to 6.85±0.05. Take 3mL of the above bacterial suspension, add it to 1mL of xylene organic solvent, mix it on a vortex oscillator for 30s, and then place the suspension at 37℃ for 30min. After the incubation time is over, take 200mL of the upper solution to determine its OD6oom value. The formula for calculating the hydrophobicity of the bacterial surface is:

[0078]

[0079] Among them, A0 represents the OD of the initial bacterial suspension 600nm Value, At represents the OD of the upper solution after incubation for 30 min 600nm value.

[0080] The results are shown in Table 4. The hydrophobicity of BA GYJC-001 in xylene can reach 85%, which has high hydrophobicity.

[0081] Table 4. Hydrophobicity test results

[0082] time result(%) 30min 85.78±0.44

[0083]

Bacterial self-agglutination ability

[0084] Take the activated strain to be tested, culture it anaerobically at 39℃ overnight, centrifuge it at 4000rpm for 20min, discard the supernatant, and repeat this step twice in PBS buffer. Centrifuge it at 4000rpm for 20min again, discard the supernatant, and resuspend the precipitate in PBS solution so that its absorbance at 600nm reaches 0.5±0.02. Vortex 1mL of bacterial suspension with a vortex mixer for 10s, incubate it at 39℃, and carefully aspirate the supernatant at 12h to measure its absorbance at 600nm. The self-aggregation formula is:

[0085]

[0086] Where T0 represents the initial bacterial suspension OD600nm Value, T12 represents OD after 12h incubation 600nm value.

[0087] The results are shown in Table 5. The 12h self-agglutination ability of BA GYJC-001 is moderate, and the self-agglutination rate can reach 55%. The above experimental results show that GYJC-001 has strong acid and alkali resistance and self-agglutination ability, suggesting that GYJC-001 has strong intestinal colonization potential.

[0088] Table 5 Self-agglutination test results

[0089] time result(%) 12h 55.16±0.50

[0090]

Bacteriostatic ability of strains

[0091] The selected colonies were activated and placed in 50 mL of MRS liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L sodium acetate, 2 g / L potassium dihydrogen phosphate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.2 g / L manganese sulfate, 1.0 mL / L Tween 80, 20 g / L glucose, and the rest was double distilled water; the pH value was adjusted to 6.2-6.6 with glacial acetic acid and sterilized at 121 °C for 20 minutes) for culture. The initial inoculation concentration was 1.0 × 10 8 CFU / mL, and each bacterial solution was adjusted to 1.0×10 10 The bacterial solution was centrifuged at 4000 rpm for 10 min, and the culture supernatant (CFS) was filtered through a 0.22 μm sterile filter and aliquoted for later use.

[0092] The antibacterial experiment was carried out by the Oxford cup double-layer plate diffusion method. First, the Escherichia coli or Salmonella suspension was inoculated into 20mL LB solid culture medium cooled to 45℃-55℃ at a 1% (v / v) inoculation amount and mixed thoroughly. After it solidified naturally, the Oxford cup was taken out vertically with sterile tweezers, and 200μL of BA GYJC-001 CFS, gentamicin (GM), MRS culture medium and PBS were added to the wells, respectively, with GM as a positive control. After the plate was placed in a 37℃ constant temperature incubator for 48h, the diameter of the inhibition zone measured by the vernier caliper was expressed as the average value.

[0093] like Figure 2 , 3 As shown in Figures 4 and 5, GYJC-001 has significant inhibitory ability (P<0.001) against cat-origin Salmonella Enteritidis Dublin (SD), cat-origin Salmonella Typhimurium (ST) and cat-origin Escherichia coli (EC), and the inhibitory ability is the same as that of gentamicin, with the average diameters of the inhibition zones reaching 19 mm, 18 mm and 20 mm, respectively.

[0094]

Antioxidant and anti-inflammatory ability of strains

[0095] (1) Cell culture

[0096] Feline renal epithelial cell (CRFK) cell line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). CRFK cells were cultured in a humidified 5% CO2 incubator at 37°C using DMEM (HyClone, UT, USA) medium containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NE, USA) and 1% antibiotics (100 mg / mL streptomycin and 100 U / mL penicillin).

[0097] (2) Effects of strains on the antioxidant function of CRFK cells

[0098] CRFK cells were collected at 5×10 5 The cells were inoculated at a density of 10 cells / well in a 12-well cell culture plate (Corning LifeScience, MA, USA), and a control group, a BA group, and a BA postbiotic group were set up, specifically:

[0099] BA live bacteria group (BA active ), BA inactivated bacteria group (BA inactive ) and BA culture supernatant group (BA CFS ),

[0100] Control group: No Bifidobacterium animalis GYJC-001 and its postbiotic substances were added, and only DMEM medium was used for 12 h and the supernatant was collected;

[0101] BA live bacteria group (BA active ): Add 10 8 CFU / mL Bifidobacterium animalis GYJC-001 was treated, and the supernatant was collected after culturing with DMEM medium for 12 h;

[0102] BA inactivated bacteria group (BA inactive ): Add 10 8 CFU / mL animal Bifidobacterium GYJC-001 was treated with inactivated bacteria, and the supernatant was collected after culturing with DMEM medium for 12 h;

[0103] BA culture supernatant group (BA CFS ): Add 10 8 The sterile culture supernatant of CFU / mL animal Bifidobacterium GYJC-001 was treated and the supernatant was collected after culturing with DMEM medium for 12 hours.

[0104] Each group had 3 replicate wells. The cells were first incubated at 37°C in a humidified 5% CO2 environment until the logarithmic growth phase, and then the cells treated with BA were cultured for 12 hours and the supernatant was collected. T-SOD, CAT, T-AOC, GSH-PX, and MDA kits (Nanjing Jiancheng) were used to detect the above indicators in the cell culture medium according to the instructions.

[0105] The results are as follows Figure 5 As shown, BA live bacteria and its postbiotics can significantly improve the antioxidant capacity of CRFK cells (P<0.001).

[0106] (3) Effects of strains on LPS-induced inflammatory factors in CRFK cells

[0107] CRFK cells were collected at 5×10 5 The cells were inoculated at a density of 10 cells / well in a 12-well cell culture plate (Corning LifeScience, MA, USA), and a control group, an LPS group (lipopolysaccharide) and a test strain group were set up, specifically:

[0108] Control group: No animal Bifidobacterium GYJC-001 was added, and the cells were cultured with DMEM medium for 12 hours, and the supernatant was discarded. The cells were washed three times with sterile PBS, and 1 mL of DMEM medium was added to continue culturing for 6 hours before collecting the samples.

[0109] LPS group: No Bifidobacterium animalis GYJC-001 was added, the cells were cultured with DMEM medium for 12 h, the supernatant was discarded, the cells were washed three times with sterile PBS, and 1 mL of DMEM medium containing LPS (1 μg / mL; Sigma-Aldrich, MO, USA) was added for 6 h before the samples were collected;

[0110] Test strain group: Use 10 8 CFU / mL inactivated strains (BA inactive ), live bacteria (BA active ) and its culture supernatant (BA CFS ) were added with 1 mL of DMEM medium containing LPS (1 μg / mL) and the supernatant was discarded after 12 h of treatment. The cells were washed three times with sterile PBS and 1 mL of DMEM medium containing LPS (1 μg / mL) was added and treated for 6 h before collecting the samples;

[0111] Each group had 3 replicate wells. The cells were first incubated at 37°C in a humidified 5% CO2 atmosphere until the logarithmic growth phase. The culture medium was then discarded and the cells were washed three times with sterile PBS. 1 mL of DMEM culture medium was added to the control group and LPS group, while 1 mL of 10 8CFU / mL inactivated DMEM medium of the strain to be tested. After pretreatment for 12 h, the supernatant was discarded, and the cells were washed three times with sterile PBS. The control group was added with 1 mL of DMEM medium, while the LPS group and BA and its postbiotic group were added with 1 mL of DMEM medium containing LPS (1 μg / mL) and treated for 6 h before collecting the cell supernatant samples.

[0112] IL-1β, IL-18, IFN-γ, IL-6, IL-4, IL-10, and IL-8 in the cell culture medium were detected according to the instructions of the ELISA kit (Raybiotech).

[0113] The results are as follows Figure 6 As shown in the results, compared with the control group, LPS induced CRFK cells, leading to upregulation of the expression of proinflammatory cytokines IL-1β, IL-18, IFN-γ, IL-6, and IL-8 (P<0.01). However, pretreatment of CRFK with animal Bifidobacterium GYJC-001 significantly alleviated the increased expression of inflammatory cytokines caused by LPS (P<0.01). At the same time, pretreatment of CRFK with animal Bifidobacterium GYJC-001 significantly increased the decrease in the level of anti-inflammatory factors caused by LPS (P<0.01).

[0114] The above results show that animal Bifidobacterium GYJC-001 can not only improve the antioxidant capacity of cells, but also alleviate inflammation by inhibiting the pro-inflammatory factors produced by CRFK induced by LPS. It suggests that animal Bifidobacterium GYJC-001 can enhance the immune function of cells by improving antioxidant capacity and relieving inflammation.

[0115] The present invention provides a method for preparing an animal bifidobacterium fermentation agent, comprising the following steps:

[0116] (1) Inoculate Bifidobacterium animalis GYJC-001 in MRS liquid culture medium and culture at 39°C for 12-18 hours for activation to obtain a culture medium containing 1×10 Bifidobacterium animalis GYJC-001. 8 -1×10 9 CFU / mL activated culture;

[0117] (2) The above-mentioned Bifidobacterium animalis GYJC-001 was added at 1×10 8 -1×10 9The activated culture was inoculated into MRS liquid culture medium at 2% CFU / mL, cultured for 24-48 hours, centrifuged at 4°C and 4000 r / min for 10-15 minutes, and the precipitate was collected and washed three times with sterile water to obtain animal Bifidobacterium fermentation agent.

[0118] The MRS liquid culture medium comprises: casein peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 5.0 g, sodium acetate 5.0 g, diammonium citrate 2.0 g, Tween 80 1.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4.H2O 0.05 g, CaCO3 20.0 g, agar 15.0 g, ultrapure water 1.0 L, pH = 6.5, and sterilization conditions: 121° C., 15 min.

[0119] The MRS liquid culture medium may also be replaced by a solid culture medium, a liquid culture medium or a semi-solid culture medium.

[0120] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A cat-derived bifidobacterium, characterized in that: Named Bifidobacterium animalis GYJC-001, the strain was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number CCTCCNO: M20241621.

2. The cat-derived Bifidobacterium according to claim 1, characterized in that The 16SrRNA gene sequence of the Bifidobacterium animalis GYJC-001 is shown in SEQ ID NO.

1.

3. The cat-derived Bifidobacterium according to claim 1, characterized in that The survival rate of the Bifidobacterium animalis GYJC-001 after being treated with a culture solution with a pH value of 2.0 to 3.0 for 3 hours is above 90%, and the self-aggregation rate after being treated for 12 hours is above 50%.

4. The cat-derived Bifidobacterium according to claim 1, characterized in that The Bifidobacterium animalis GYJC-001 is separated from lactic acid bacteria in the feces of Chinese garden cats.

5. The cat-derived Bifidobacterium according to claim 1, characterized in that The Bifidobacterium animalis GYJC-001 has acid resistance, alkali resistance, self-aggregation ability and intestinal colonization ability.

6. The cat-derived Bifidobacterium according to claim 1, characterized in that The Bifidobacterium animalis GYJC-001 has the ability to inhibit the growth of pathogenic microorganisms.

7. The cat-derived Bifidobacterium according to claim 1, characterized in that The Bifidobacterium animalis GYJC-001 has the ability to inhibit the production of inflammatory factors by cat kidney cells induced by LPS in vitro and improve the antioxidant capacity of cat kidney cells.

8. An animal bifidobacterium postbiotic, characterized in that: The animal Bifidobacterium postbiotic is the fermentation agent, culture, bacterial suspension or lysate of cat-derived animal Bifidobacterium as described in any one of claims 1 to 7.

9. A composition, characterized in that The method comprises one or more of the cat-derived Bifidobacterium, its culture, its bacterial suspension and its lysate as described in any one of claims 1 to 7.

10. Use of the cat-derived Bifidobacterium according to any one of claims 1 to 7 in the preparation of a medicament for preventing and / or treating intestinal diseases.

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