A strain of Enterococcus faecium from dog, Kimate-X, and its application

Abstract

The present invention discloses a strain of Enterococcus faecium ( Enterococcus feces )Kimate‑X and its applications ， The Enterococcus faecium ( Enterococcus faecium )Kmiate‑X was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC No. 29389. The strain was isolated and identified from the feces of healthy dogs. The bacteria can be used as a dog food additive and has the potential for anti-inflammatory and anti-stress effects. It provides a new idea for maintaining dog health and also provides a reliable backup strain for canine microecological preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a canine-derived Enterococcus faecium Kimate-X and an application thereof. Background Art

[0002] Dogs play an increasingly important role in modern society. Whether as family members, service animals or professional working partners, their presence enriches human life and takes on a variety of responsibilities and roles. However, like humans, dogs often face various stresses and challenges, especially during transportation. Whether it is long-distance travel, participating in competitions or relocating to a new home, these situations may cause discomfort and stress reactions to dogs.

[0003] The stress and discomfort that dogs experience during transportation is not only physical, psychological stress is also a serious problem. Anxiety, fear and abnormal behavior often accompany the transportation process, which not only affects the physical and mental health of dogs, but may also affect their adaptation and performance at the destination. Therefore, it is crucial to find a way to reduce the stress of dog transportation, which will not only help improve the quality of life of dogs, but also help improve their interaction and cooperation with humans.

[0004] In this context, our research focuses on finding an innovative solution to alleviate the stress response of dogs during transportation. By exploring the relationship between intestinal microorganisms and animal health, we found that probiotics may become a promising coping strategy. Therefore, this study aims to develop a specific probiotic that can reduce the stress response during transportation by regulating the composition of canine intestinal microorganisms, thereby improving the transportation experience and health level of dogs.

[0005] So far, there have been no reports on the effectiveness of canine-derived Enterococcus faecium in relieving stress in dogs. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Enterococcus faecium Kimate-X which is acid-resistant, bile-resistant and can effectively alleviate the damage caused by transportation stress to dogs.

[0007] The technical problem that the present invention also aims to solve is to provide a probiotic preparation containing the Enterococcus faecium Kimate-X.

[0008] The technical problem that the present invention also aims to solve is to provide the use of Enterococcus faecium Kimate-X or the probiotic preparation in preparing feed and medicine for alleviating the oxidative stress level of the body.

[0009] The final technical problem to be solved by the present invention is to provide the use of the Enterococcus faecium Kimate-X or the probiotic preparation in the preparation of feed and medicine for preventing or alleviating canine transportation stress. The present invention will bring new perspectives and solutions to the field of animal health care.

[0010] Technical solution: In order to solve the above technical problems, the present invention provides a strain of Enterococcus faecium Kimate-X, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC No.29389.

[0011] The invention also provides a probiotic preparation, which contains the Enterococcus faecium Kimate-X.

[0012] The present invention also provides the use of the Enterococcus faecium Kimate-X or the probiotic preparation in preparing feed and medicine for alleviating the oxidative stress level of the body.

[0013] The present invention also provides the use of the Enterococcus faecium Kimate-X or the probiotic preparation in preparing feed and medicine for promoting the diversity and uniformity of canine intestinal microorganisms.

[0014] The present invention also provides the use of the Enterococcus faecium Kimate-X or the probiotic preparation in preparing feed and medicine for improving the level of short-chain fatty acids in the canine intestine.

[0015] The application includes improving the body's antioxidant index, specifically including improving the expression level of SOD and / or CAT.

[0016] The application includes reducing the body's oxidative stress index MDA and inflammatory index TNFα.

[0017] The present invention also provides the use of the Enterococcus faecium Kimate-X or the probiotic preparation in preparing feed and medicine for preventing or alleviating canine transportation stress.

[0018] The application specifically includes preventing emergency reactions caused during transportation by increasing the anti-oxidative stress index serum SOD and reducing the oxidative stress index MDA and the inflammatory index TNFα.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the Enterococcus faecium Kimate-X of the present invention has the properties of acid resistance and bile salt resistance, and can effectively alleviate the damage caused by transportation stress to dogs. The bacteria can be used as a dog food additive, has the ability to relieve dog stress, provides a new idea for maintaining dog growth, optimizing intestinal flora and alleviating stress damage, and also provides a reliable backup strain for dog-derived microecological preparations. The present invention is suitable for feed mills and large-scale dog farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the cell experiment results of the "Canine Faecium Enterococcus Kimate-X" of the present invention.

[0021] Figure 2 This is the animal experiment serum test result of the "Canine Faecium Enterococcus Kimate-X" of the present invention.

[0022] Figure 3 These are the results of fecal microbiome analysis of the animal experiment on the “Canine Faecium Enterococcus Kimate-X” of the present invention.

[0023] Figure 4 This is the test result of short-chain fatty acids in feces of the animal experiment “Canine Faecium Enterococcus Kimate-X” of the present invention. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0025] Example 1 Acquisition, identification and biological characteristics of Enterococcus faecium Kimate-X

[0026] 1. Strain Isolation

[0027] Prepare MRS culture medium. Preparation method: Add an appropriate amount of MRS culture medium (peptone: 10.0g / L, beef extract: 10.0g / L, yeast extract: 5.0g / L, glucose: 20.0g / L, ammonium citrate: 2.0g / L, magnesium sulfate: 0.1g / L, dipotassium hydrogen phosphate: 2.0g / L, manganese sulfate: 0.05g / L, sodium acetate: 5.0g / L, Tween 80: 1.0g / L, and distilled water to 1 liter). Pour the culture medium into a sterile culture dish, seal and sterilize. Take 0.5g of dog feces sample and add it to a sterile tube, suspend it with 5mL of sterile 0.9% saline, mix it with a vortex mixer to form a suspension, filter it, and dilute the filtrate with sterile saline for a multiple of 10 -5 , 10-6 , 10 -7 Gradient, each 100 μL was drawn, inoculated onto the MRS medium plate by the plate method (spreading the diluted sample on the surface of the culture medium), and incubated at 37°C incubator for 24 hours. Observe the colonies formed on the culture dish, select a single colony, and use a sterile pipette to culture the dish, incubate at 37°C for 24 hours. Pick a single colony and inoculate it into 10 mL of MRS liquid culture medium and culture it at 37°C for 20 hours to obtain a pure culture solution of a single lactic acid bacteria.

[0028] 2. Molecular biological identification of strains

[0029] The obtained pure culture of lactic acid bacteria was streaked onto MRS solid plates with an inoculation needle and incubated at 37°C for 12 hours. The single colony on the plate was picked up with a pipette tip. The purified bacterial single colony was directly used as a DNA template to amplify the 16S rRNA gene of the strain. The bacterial identification 16S rDNA universal primers 27F: 5'-AGAGTTTGA TCMTGGCTCAG-3' and 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' were used for PCR amplification, which were synthesized by Shanghai Shenggong Biological Co., Ltd. The PCR amplification system was: 25μL 2×Taq PCR Master Mix; 19μL ddH2O; 2μL forward primer 27F (25μmol / L), 2.0μL reverse primer 1492R (25μmol / L) and 2μL DNA template. The amplification conditions were 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 7 min, and 4°C to terminate the reaction. The PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After obtaining the 16S rRNA gene of the strain, the sequence was compared with the NCBI database to determine that the isolated bacteria was Enterococcus faecium, which is the Enterococcus faecium Kimate-X of the present invention. The strain was deposited in the China General Microbiological Culture Collection Center on December 25, 2023, with the deposit number: CGMCC No. 29389.

[0030] 3. Acid resistance test of Enterococcus faecium Kmiate-X strain

[0031] Probiotics acid resistance test: Kimate-X (1×10 7CFU) bacterial solution, each with 10 mL, centrifuged and discarded the supernatant to obtain bacterial precipitate. Add 10 mL of sterile PBS (sodium chloride (NaCl): 8.0 g sodium dihydrogen phosphate (NaH2PO4·H2O): 0.144 g dihydrogen phosphate (Na2HPO4·2H2O): 1.44 g potassium chloride (KCl): 0.2 g distilled water to 1 liter, adjust the pH to 7.4) to each portion, rinse and mix, and wash away the residual culture medium. Then centrifuge and discard the supernatant to obtain bacterial precipitate. Add 10 ml of sterile PBS with a pH value of 2 to three of the bacterial precipitates, and add 10 ml of untreated sterile PBS to the other three portions. The six bacterial solutions were incubated in an incubator at 37°C. Two bacterial solutions were taken out and centrifuged every 1 hour, and the supernatant was discarded. Each was rinsed and suspended with 10 mL of sterile PBS to wash away the residual treatment solution. The two tubes of bacterial solution were diluted and applied, and the dilution gradient was 10 6 , 10 7 , 10 8 After incubation at 37°C for 12 hours, the colonies were counted and the bacterial concentration of the culture was calculated. The survival rate of bacteria at pH 2 was calculated using the following formula: survival rate of bacteria at pH 2 = (pH = 2 concentration of the culture after PBS treatment / concentration of the culture after ordinary PBS treatment)%. Bifidobacterium BB-12, a commonly used probiotic strain for dogs, was selected as a reference strain and the acid resistance test was performed using the same method.

[0032] Through the acid resistance test, the acid resistance test results are shown in Table 1. The survival rate of Enterococcus faecium Kimate-X exposed to a pH 2 environment for 3 hours is still higher than 60%, which is significantly higher than the reference strain Bifidobacterium BB-12, and has strong acid resistance.

[0033] Table 1 Results of acid resistance test of Kimate-X strain

[0034]

[0035] 4. Bile salt detection of Enterococcus faecium Kimate-X strain

[0036] The test steps are basically the same as those of the probiotic acid resistance test, except that the pH=2 PBS needs to be replaced with 0.3% swine bile salt concentration PBS. The formula for calculating the bacterial survival rate under 0.3% swine bile salt saline treatment is: bacterial survival rate under 0.3% bile salt conditions = (bacterial solution concentration after 0.3% swine bile salt PBS treatment / bacterial solution concentration after ordinary PBS treatment)%.

[0037] Through the bile salt resistance test, the bile salt resistance test results are shown in Table 2. The survival rate of Enterococcus faecium Kimate-X after being exposed to a high concentration of bile salt environment for 3 hours is still higher than 45%, which is similar to the survival rate of the reference strain Bifidobacterium BB-12, with no significant difference, indicating that it has strong bile salt resistance.

[0038] Table 2 Results of Kimate-X strain bile salt tolerance test

[0039]

[0040] Example 2 Study on the effect of Enterococcus faecium Kimate-X on alleviating oxidative stress in RAW264.7 cells in vitro

[0041] RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics and incubated in a constant temperature incubator at 37°C with 5% CO2. In the case of well-growing RAW 264.7 cells, the cell density was 5 × 10 5 / mL, and treated with 500ng / mL LPS to induce oxidative stress. While inducing oxidative stress, E. faecium Kimate-X was inoculated with 1×10 5 The inoculum of CFU was added to the culture medium containing RAW 264.7 cells after the above induction, and experimental and control groups were set up, including the oxidative stress model group: RAW 264.7 cells were exposed to LPS treatment; probiotic treatment group: RAW264.7 cells were exposed to probiotic treatment under the oxidative stress model; control group: RAW264.7 cells were cultured under normal conditions without LPS or probiotic treatment. After 12 hours of culture, the cell supernatant was collected to detect oxidative stress and inflammation-related indicators.

[0042] The experimental results of Kimate-X strain alleviating oxidative stress in RAW264.7 cells in vitro are as follows Figure 1 As shown, after LPS modeling, the antioxidant indicators SOD and CAT in the probiotic intervention group were significantly higher than those in the LPS model group, the oxidative stress indicator MDA in the probiotic intervention group was significantly lower than that in the LPS model group, and the inflammatory indicator TNFα in the probiotic intervention group was significantly lower than that in the LPS model group.

[0043] Example 3 Study on the Effect of Enterococcus faecium Kimate-X on Relieving Transport Stress in Dogs

[0044] Sixteen 3-month-old beagle dogs of similar weight were randomly divided into two groups, a probiotic intervention group and a control group, with 8 dogs in each group. Both groups were fed the same commercial dog food. During the experiment, there was no restriction on food and water. The probiotic intervention group was additionally fed 2×10 9CFU of Enterococcus faecium Kmiate-X. Transport stress was performed after 7 weeks. All dogs were placed in a closed truck and transported at 60 km / h for 3 hours. The temperature in the truck was controlled at 23 degrees Celsius and the relative humidity was 60%. The transportation started at 9:00 am, including urban roads and highways. After the transportation, 3 ml of blood was immediately collected using a vacuum blood collection tube. After the collected blood was allowed to stand at 4°C for 1 hour, it was centrifuged at 4000 rpm for 10 minutes to collect serum for the detection of stress and inflammatory indicators. At the same time, fresh feces of the dogs (within 15 minutes after defecation) were collected, packaged and stored at -80°C for fecal short-chain fatty acids (SCFA) and microbiota detection and analysis.

[0045] The experimental results of Enterococcus faecium Kmiate-X in alleviating transportation stress in dogs are as follows Figure 2 As shown, changes in multiple physiological indicators of the control group and the probiotic intervention group (Kimate-X) under stress conditions. Figure 2 A shows that the cortisol level in the probiotic intervention group was significantly lower than that in the control group, indicating that probiotics can effectively reduce the increase in cortisol caused by stress. Figure 2 B shows that the SOD activity of the probiotic intervention group was significantly higher than that of the control group, indicating that probiotics can enhance antioxidant capacity and reduce oxidative stress. Figure 2 C showed that there was no significant difference in CAT activity between the two groups, indicating that probiotics had little effect on catalase activity. Figure 2 D shows that the MDA level in the probiotic intervention group was significantly higher than that in the control group, suggesting that probiotics can alleviate stress damage. Figure 2 E showed that the TNF-α level in the probiotic intervention group was significantly lower than that in the control group, indicating that probiotics can reduce stress response by inhibiting inflammatory factors. In summary, probiotic intervention has a significant alleviating effect on the stress response of dogs in many aspects.

[0046] Fecal microbiota diversity analyses (including alpha diversity and beta diversity) were performed and visualized using the vegan and ggplot2 packages in R (version 4.2.1). Specifically, alpha diversity indices such as the Shannon index and the Simpson index were calculated using the vegan package. Differences in alpha diversity indices between groups were tested using the Wilcoxon rank-sum test. Principal coordinates analysis based on Bray-Curtis distances was used to examine differences in microbiota structure. Differences in PCoA ordination between groups were tested using permutational multivariate analysis of variance.

[0047] Figure 3 The diversity and composition analysis of fecal microbiota of dogs in the control (Con) and probiotic intervention groups (Kimate-X) are presented. Figure 3 A and Figure 3 B shows the Shannon index and Simpson index, respectively. The diversity index of the probiotic intervention group was significantly higher than that of the control group, indicating that probiotic intervention improved the diversity of intestinal microbiota. Figure 3 Principal coordinate analysis (PCoA) of C showed that the microbiota structures of the two groups were clearly separated, indicating that probiotic intervention significantly changed the composition of the intestinal microbiota. Figure 3 D shows the Bray-Curtis distance. The distance of the probiotic intervention group was significantly lower than that of the control group, indicating that the microbiota in the probiotic intervention group were more similar, further supporting the regulatory effect of probiotics on intestinal microbiota. In summary, probiotic intervention has a significant effect in improving the diversity of canine intestinal microbiota and optimizing community structure.

[0048] Fecal samples of dogs in the control group (Con) and probiotic intervention group (Kimate-X) were sent to Zhongke Xinsheng Biotechnology Co., Ltd. (Shanghai) for short-chain fatty acid analysis. The specific method is as follows: After the sample is thawed on ice, an appropriate amount is placed in a 2mL centrifuge tube. Then 50μL of 20% phosphoric acid is added for resuspending, and 4-methylvaleric acid with a final concentration of 500μM is added as an internal standard and mixed thoroughly for 2 minutes. The mixture is centrifuged at 14,000g for 20 minutes, and the supernatant is transferred to an injection bottle for GC-MS analysis. Take 1μL of sample with a split ratio of 10:1. The sample is separated by an Agilent DB-FFAP capillary column (30m x 250μmx 0.25μm) gas chromatography system. The temperature program is as follows: the initial temperature is 90℃, and it is increased to 160℃ at a rate of 10℃ / min; then it is increased to 240℃ at a rate of 40℃ / min and maintained for 5 minutes. Helium was used as the carrier gas with a flow rate of 1.0 mL / min. In the sample sequence, a QC sample was set every certain number of experimental samples to detect and evaluate the stability and repeatability of the system.

[0049] Figure 4 The levels of various short-chain fatty acids (SCFAs) in the feces of dogs in the control group (Con) and the probiotic intervention group (Kimate-X) were presented. The results showed that the levels of acetate, propionate, and butyrate in the probiotic intervention group were significantly higher than those in the control group, indicating that probiotic intervention significantly increased the production of these major SCFAs. These fatty acids play an important role in maintaining intestinal health and overall metabolism. Although there was little difference between the two groups in less abundant SCFAs (such as valeric acid, hexanoic acid, heptanoic acid, and octanoic acid), the overall results support the potential benefits of probiotics in enhancing intestinal health.

Claims

1. A strain of Enterococcus faecium ( Enterococcus faecium )Kimate-X ， Characterized in that the Enterococcus faecium ( Enterococcus faecium )Kmiate-X was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC No. 29389.

2. A probiotic preparation, characterized in that: It contains the Enterococcus faecium according to claim 1 ( Enterococcus faecium ) Kimate-X.

3. The Enterococcus faecium according to claim 1 ( Enterococcus faecium ) Use of Kimate-X or the probiotic preparation according to claim 2 in the preparation of feed for alleviating the level of oxidative stress in the body.

4. The use according to claim 3, characterized in that: The application includes improving the body's antioxidant index, specifically including improving the expression level of SOD and / or CAT.

5. The use according to claim 3, characterized in that: The application includes reducing the body's oxidative stress index MDA and inflammation index TNF-α.

6. The Enterococcus faecium according to claim 1 ( Enterococcus faecium ) Use of Kimate-X or the probiotic preparation according to claim 2 in the preparation of feed for promoting the diversity and uniformity of canine intestinal microorganisms.

7. The Enterococcus faecium according to claim 1 ( Enterococcus faecium ) Use of Kimate-X or the probiotic preparation according to claim 2 in the preparation of feed for increasing the level of short-chain fatty acids in the canine intestine, wherein the short-chain fatty acids are one or more of acetic acid, propionic acid or butyric acid.

8. The Enterococcus faecium of claim 1 ( Enterococcus faecium ) Use of Kimate-X or the probiotic preparation according to claim 2 in the preparation of feed and medicine for preventing or alleviating canine transportation stress.

9. The use according to claim 8, characterized in that: The application specifically includes preventing stress reactions caused during transportation by increasing the anti-oxidative stress index serum SOD and reducing the oxidative stress index MDA and the inflammatory index TNF-α.

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