Dog-derived enterococcus faecium and application thereof

Abstract

The enterococcus faecium LBEF-01 is separated and screened from healthy dog feces, the strain is preserved in the Guangdong Microbial Culture Collection Center on August 15, 2024, the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC NO.65013. The enterococcus faecium LBEF-01 has the advantages that the enterococcus faecium LBEF-01 can be used for preparing the enterococcus faecium LBEF-01; in-vitro research shows that the strain has the advantages of relatively high acid resistance and intestinal juice resistance, can tolerate high-concentration bile salt, has bile salt hydrolase activity and has the capacity of inhibiting intestinal pathogenic bacteria. In-vivo studies show that the strain enhances excretion of total bile acid in excrement and reduces cholesterol content in blood by promoting bile acid metabolism. The strain can improve bile acid in-vivo circulation of pets, and can improve liver-derived tear stain symptoms of pets after being taken.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a canine-derived Enterococcus faecium LBEF-01 and an application thereof. Background Art

[0002] As the number of pet-raising families increases year by year, pet health issues are becoming a focus of attention. Pet tear stains are a common pet eye disease, and the cause of their formation is mostly porphyrin staining. Porphyrin is an iron-containing molecule and an important substance that constitutes hemoglobin, myoglobin, and cytochrome. Generally, porphyrins formed by metabolism are excreted in the form of feces and urine, and are also excreted through tears and saliva of dogs and cats.

[0003] The liver is the main organ that regulates the anabolism of porphyrins and their precursors in dogs and cats, and is also the key organ affected by abnormal porphyrin metabolism. Protoporphyrin synthesized in the body is mainly secreted into bile by the liver and excreted from the bile duct into the enterohepatic circulation. Bile acid is a class of molecules with the same structure that are synthesized from cholesterol in the liver and modified by intestinal flora. In the intestinal cavity, bile acid plays the role of a lipid solubilizer and is essential for lipid absorption. In addition to dissolving lipids, bile acid also plays other important physiological roles, regulating liver and intestinal function. When the metabolism of bile acid in the pet's body is abnormal, the enterohepatic circulation of bile acid is blocked, the bile acid content in the feces is reduced, and the porphyrin excreted through the feces is reduced. Porphyrin is excreted through tears to form hepatic tear stains. Hepatic tear stains not only affect the appearance of pets, but also affect the health of pets, and may induce eye inflammation, etc. Therefore, the tear stain problem of pets should be taken seriously.

[0004] Studies have shown that intestinal flora is one of the important factors affecting bile acid metabolism, and the composition of intestinal flora controls bile acid synthesis in the host and the enterohepatic circulation of bile acid. At present, the prevention and treatment of pet tear marks are mostly through supplementing bile acid solution and supplementing Chinese medicine extracts. Although this method can improve the symptoms of pet tear marks, it needs to be taken for a long time, and there is a certain risk of recurrence of pet hepatic tear marks after stopping taking it. At present, there are few studies on whether probiotics can improve the symptoms of pet hepatic tear marks by improving bile acid metabolism. Patent application CN113143971A discloses the use of bile acid composite bacterial agents in the preparation of preparations for preventing and treating pet tear marks, and specifically discloses a composite bacterial agent composed of Parabacteroides dissimilar LCG-06 derived from the pig intestine and bile acid. However, the patent application does not explain whether Parabacteroides dissimilar LCG-06 can further enhance bile acid metabolism after compounding with bile acid, nor does it explain whether taking the probiotics alone can improve pet hepatic tear marks. Summary of the invention

[0005] In view of the above situation, the present invention provides an Enterococcus faecium that can improve pets' hepatic tear stains.

[0006] The present invention isolates and screens a strain of Enterococcus faecium from healthy dog ​​feces, the strain is named Enterococcus faecium LBEF-01, and is preserved in Guangdong Microbial Culture Collection Center, the address of the collection center is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, the preservation date is August 15, 2024, the preservation number is GDMCC No: 65013, and the classification name is Enterococcus faecium.

[0007] As one of the embodiments, the 16S rDNA of the Enterococcus faecium LBEF-01 is as shown in SEQ ID NO:1.

[0008] The present invention provides the use of Enterococcus faecium LBEF-01 as a live bacterium in a probiotic preparation, and the preparation is in the form of a lyophilized powder preparation or a bacterial suspension preparation, preferably a lyophilized powder preparation.

[0009] The invention provides a probiotic freeze-dried powder preparation, comprising live Enterococcus faecium LBEF-01 bacterial bodies and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises one or more of sugars, alcohols and composite protective agents.

[0010] As one of the preferred embodiments of the present invention, the lyophilization protective agent comprises sugars, alcohols and composite protective agents.

[0011] Furthermore, the sugar is selected from at least one of lactose, glucose, trehalose, dextran, xylooligosaccharides or fructo-oligosaccharides, preferably fructo-oligosaccharides; the alcohol is selected from at least one of glycerol, mannitol or sorbitol, preferably glycerol; the composite protective agent is selected from at least one of skimmed milk powder, maltodextrin or soluble starch, preferably skimmed milk powder.

[0012] Furthermore, the probiotic freeze-dried powder preparation comprises, by weight percentage, live Enterococcus faecium LBEF-01 bacteria and 10%-20% skim milk powder, 1.5%-2% glycerol, 3%-5% oligofructose, and the remainder is sterile water.

[0013] As one of the preferred embodiments of the present invention, the probiotic freeze-dried powder preparation comprises live Enterococcus faecium LBEF-01 bacteria and 15% skim milk powder, 2% glycerol, 5% oligofructose, and the balance is sterile water.

[0014] The present invention also provides the use of the above-mentioned Enterococcus faecium LBEF-01 or the above-mentioned probiotic freeze-dried powder preparation in the preparation of pet products for improving hepatic tear marks caused by abnormal enterohepatic circulation in pets, bacterial diarrhea or related diseases caused by intestinal flora imbalance.

[0015] Furthermore, the pet products include pet medicines, pet food and pet health products.

[0016] Furthermore, the pets include pet dogs and pet cats.

[0017] The present invention provides a method for preparing the above-mentioned probiotic freeze-dried powder preparation, and the method is as follows:

[0018] Enterococcus faecium LBEF-01 colonies are inoculated into a BHI liquid culture medium or an MRS liquid culture medium, and statically cultured at 30-37° C. (preferably 37° C.) for 14 h-16 h to obtain an activated bacterial solution; the activated bacterial solution is inoculated into a BHI liquid culture medium or an MRS liquid culture medium at a ratio of 1%-2%, and cultured at 30-37° C. (preferably 37° C.); the fermentation liquid is centrifuged to collect bacterial bodies, the bacterial bodies are resuspended and washed with sterile water, and then a lyophilization protective agent is added; and probiotic lyophilized powder is obtained after low-temperature freeze-drying.

[0019] The present invention has the following technical effects:

[0020] 1. In vitro studies on Enterococcus faecium LBEF-01 have shown that the strain has good acid and intestinal fluid resistance, and it is also found that the strain can tolerate higher concentrations of bile salts; the strain has bile salt hydrolase (BSH) activity and can be used in medicines or foods; the strain can inhibit Salmonella typhimurium, Shigella and Escherichia coli, and has potential as a probiotic. In vivo studies have shown that the strain has the ability to improve bile acid metabolism, enhance the enterohepatic circulation of bile acid, increase the bile acid concentration in feces and reduce the cholesterol concentration in the blood, and has a certain effect on improving the symptoms of hepatic tear stains in pets.

[0021] 2. The Enterococcus faecium LBEF-01 of the present invention is derived from healthy dog ​​feces, has good tolerance in pets, and can be used as a probiotic for pets.

[0022] 3. The experimental results of feeding Enterococcus faecium LBEF-01 to canine models showed that the preparation containing Enterococcus faecium LBEF-01 has the effect of improving pet bile acid metabolism and enhancing the enterohepatic circulation of pet bile acid, which can improve pet hepatic tear stains and can be used for related diseases caused by abnormal bile acid flora.

[0023] 4. Using the probiotic freeze-dried powder preparation formula of the present invention, the viable survival rate of Enterococcus faecium LBEF-01 is maintained at more than 60%, and can even reach 94%-100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony morphology of Enterococcus faecium LBEF-01;

[0025] Figure 2This is the Gram staining image of Enterococcus faecium LBEF-01;

[0026] Figure 3 This is the 16S rDNA phylogenetic map of Enterococcus faecium LBEF-01;

[0027] Figure 4 It is the electrophoresis fingerprint of Enterococcus faecium LBEF-01 and the control bacteria Enterococcus faecium NCIMB 10415;

[0028] Figure 5A This is the survival rate graph of Enterococcus faecium LBEF-01 in simulated gastric juice and intestinal juice;

[0029] Figure 5B is a graph showing the survival rate of Enterococcus faecium LBEF-01 at different bile salt concentrations;

[0030] Figure 6 This is a graph of bile acid content in dog feces;

[0031] Figure 7 It is a graph of cholesterol levels in dog blood;

[0032] Figure 8 is a diagram showing the tear stain removal effect of dogs after taking LBEF-01 probiotics, Fig. 8A Before taking LBEF-01 probiotics, Figure 8B After taking LBEF-01 probiotics. DETAILED DESCRIPTION

[0033] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0034] Those skilled in the art should understand that the reagents used in the following examples, unless otherwise specified, were purchased from the market.

[0035] Example 1 Isolation and Identification of Enterococcus faecium LBEF-01

[0036] Healthy dog ​​feces were collected and resuspended in an appropriate amount of sterile saline and diluted to a gradient of 10 -6 -10 -8 Then spread it on MRS solid culture medium, and after the suspension is dried, place it in an anaerobic gas production bag and culture it at 37℃ for 24h. Then streak a single colony on MRS solid culture medium for expansion culture. After anaerobic culture at 37℃ for 24h, round protrusions with smooth surface and grayish white color are obtained, such as Figure 1 Gram staining and microscopic examination confirmed that the Gram-positive enterococci were as follows. Figure 2The genomic DNA of the isolated strain was extracted using a bacterial genome extraction kit, and the 16S rDNA universal forward primer 27F (5'-AGAGTTTGA TCC TGG CTC AG-3'); universal reverse primer 1495R (5'-CTA CGG CTA CCT TGT TAC GA-3') was used for amplification and then gel-cutting and recovery. The recovered product was sequenced, and the 16S rDNA sequence of Enterococcus faecium LBEF-01 is shown in SEQ ID NO: 1. The sequencing results were submitted to NCBI for 16S rDNA sequence comparison, as shown in Figure 3 As shown, the sequence alignment results showed that the LBEF-01 strain was a canine-derived Enterococcus faecium.

[0037] SEQ ID NO:1

[0038]

[0039] Example 2 Strain Analysis of Enterococcus faecium LBEF-01

[0040] In order to further confirm whether the canine-derived Enterococcus faecium LBEF-01 is related to the animal probiotic Enterococcus faecium NCIMB 10415 strain. The enterococcal target gene ERIC-PCR method was used for analysis. The specific method is as follows: extract the total bacterial DNA from the activated strain, and add relevant reagents according to the ERIC-PCR reaction system (the total reaction volume is 25μl, add 10×PCR buffer, 0.2mmol / L dNTP, 2U Taq DNA polymerase, 0.4μmol / L primer, 2.5mmol / L MgCl2, 10ng-50ng total bacterial DNA, add sterile ddH2O to 25μL), the reaction conditions are as follows: pre-denaturation: 95℃, 5min; amplification conditions: 94℃30s, 50℃60s, 72℃60s; cycle number 35; extension: 72℃8min), the amplified product is analyzed by electrophoresis, and the electrophoresis of the fingerprint of Enterococcus faecium LBEF-01 and the control bacteria NCIMB 10415 is shown in the figure. Figure 4 shown.

[0041] Table 1 Similarity matrix of cluster analysis of fingerprints of Enterococcus faecium LBEF-01

[0042]

[0043] The results showed that the affinity between Enterococcus faecium LBEF-01 and Enterococcus faecium NCIMB 10415 was 0.68, and there were certain differences between the two strains.

[0044] Example 3 Determination of acid resistance, intestinal fluid resistance and bile salt resistance of Enterococcus faecium LBEF-01

[0045] Take an appropriate amount of activated Enterococcus faecium LBEF-01 bacterial solution and the control strain Enterococcus faecium NCIMB10415, and the viable bacteria count on the plate dilution is the initial bacterial concentration before the experiment. The bacterial solution was inoculated into artificial simulated gastric juice and intestinal juice at an inoculation rate of 10%, and the viable bacteria count was coated on the plate after incubation in a constant temperature water bath at 37°C for 3 hours, which is the bacterial concentration after the experiment. Three parallel groups were set up for each group, and the survival rate of gastric acid and intestinal juice resistance was calculated. The results are shown in Table 2 and Figure 5A As shown, the survival rate of Enterococcus faecium LBEF-01 was higher after 3 h in simulated gastric fluid and intestinal fluid.

[0046] Table 2 Average survival rate of Enterococcus faecium LBEF-01 in gastric juice and intestinal juice for 3 hours

[0047]

[0048] Take an appropriate amount of activated Enterococcus faecium LBEF-01 bacterial suspension, dilute and spread on the plate and count the number of live bacteria. Inoculate the bacterial suspension with 10% inoculum into MRS liquid culture medium with different bile salt concentrations, bile salt concentrations of 0%, 0.3%, 0.5%, 1.0%, 2.0%, 3.0%, and culture at 37℃ for 3h. After the culture is completed, count the live bacteria and calculate the survival rate.

[0049] Table 3 Survival rate of Enterococcus faecium LBEF-01 at different bile salt concentrations

[0050]

[0051] The results are shown in Table 3 and Figure 5B As shown, Enterococcus faecium LBEF-01 has good bile salt tolerance and can survive even in a high bile salt concentration environment of 3.0%.

[0052] Example 4 Detection of bile salt hydrolase of Enterococcus faecium LBEF-01

[0053] Prepare MRS solid medium containing bile salts, the preparation method is as follows: add 0.37g / L CaCl2, 0.2% sodium thioglycolate, 0.3% sodium deoxytaurocholate (TCDA) to MRS solid medium containing agar powder, sterilize after mixing. Place sterile filter paper in the medium, add 10μL bacterial solution to it, observe after anaerobic culture at 37℃ for 72h, and observe that white precipitate is generated around the filter paper, confirming that Enterococcus faecium LBEF-01 has bile salt hydrolase activity.

[0054] The activity of bile salt hydrolase of Enterococcus faecium LBEF-01 was further determined. The activated bacterial solution was centrifuged to collect the cells, and then resuspended and washed twice with sterile PBS. 10 mL of PBS was added per gram of cells to resuspend the cells, and 10 mmol / L dithiothreitol was added to prevent oxidation. After ultrasonic disruption in an ice bath, the supernatant was collected by centrifugation at 4°C and 12000 rpm to obtain the cell extract.

[0055] 180 μL 0.1 mol / L PBS solution was added to an appropriate amount of bacterial extract, and 10 μL 200 mmol / L bile salt and 10 μL 0.1 mol / L paraffin oil were added. After reacting at 37°C for 30 min, an equal volume of 0.15 mg / mL TCA was added to terminate the reaction, and the supernatant was collected by centrifugation. The catalytic activity of bile salt hydrolase was detected by the ninhydrin method, and the average value was taken after three times. The results are shown in Table 4.

[0056] Table 4 Average activity of bile salt hydrolase from Enterococcus faecium LBEF-01

[0057]

[0058] Example 5 Determination of the ability of Enterococcus faecium LBEF-01 to inhibit intestinal pathogens

[0059] Inoculate and activate Enterococcus faecium LBEF-01, collect the supernatant by centrifugation at 4°C and 5000rpm, and filter and sterilize with a 0.22μm filter for later use. The antibacterial ability of Enterococcus faecium LBEF-01 was determined by the Oxford cup agar diffusion method. An Oxford cup was placed on an LB plate with Salmonella typhimurium (GDMCC1.237), Shigella dysenteriae (GDMCC1.236), and Escherichia coli (GDMCC1.4025), and 200μL of bacterial solution was added to the Oxford cup. Negative and positive controls were set at the same time. The experiment was repeated three times and the average value was taken. The results are shown in Table 5 below, indicating that Enterococcus faecium LBEF-01 can inhibit three intestinal pathogens of Salmonella typhimurium, Shigella dysenteriae, and Escherichia coli.

[0060] Table 5 Average values ​​of inhibition zones of Enterococcus faecium LBEF-01 against intestinal pathogens

[0061]

[0062] Example 6 Screening of freeze-dried protective agent for Enterococcus faecium LBEF-01 powder

[0063] In order to further study the effect of different freeze-drying protective agent formulas on the survival rate of Enterococcus faecium LBEF-01 powder, the live bacteria of Enterococcus faecium LBEF-01 were mixed with freeze-drying protective agents of different formulas, the number of live bacteria was measured after freeze-drying, and the freeze-drying survival rate under different formulas was calculated. The specific research methods are as follows:

[0064] Inoculate a single colony of Enterococcus faecium LBEF-01 into MRS liquid culture medium, culture at 37°C for 10-14 hours, then inoculate the culture solution at a 2% ratio into an appropriate amount of fresh MRS liquid culture medium, culture at 37°C for 48 hours, and then collect the cells by centrifugation. Weigh and take an appropriate amount of cells, dissolve in sterile water, mix well, dilute 10 times to an appropriate dilution, select 3 samples with different dilutions and spread them on MRS agar medium, and culture at 37°C for 48 hours. Calculate the average number of viable cells, which is the number of viable cells before freeze-drying.

[0065] Prepare 5% different sugars (glucose, lactose, trehalose, xylo-oligosaccharides, fructo-oligosaccharides), 2% different alcohols (glycerol, mannitol, sorbitol), and 15% different composite protective agents (skim milk powder, maltodextrin, soluble starch) as a single protective agent, dissolve in sterile water and sterilize at 115°C for 20 minutes before use. Add the bacteria to the freeze-dried protective agent at a ratio of 1:50, mix well, and then freeze-dry in vacuum.

[0066] After freeze-drying, the powder was fully crushed and sampled for live bacteria counting. An appropriate amount of powder was dissolved in sterile water and mixed evenly. Three samples with different dilutions were selected and spread on MRS agar medium. After inverted culture at 37°C for 48 hours, the average number of live bacteria was calculated, which was the number of live bacteria after freeze-drying.

[0067] Freeze-drying survival rate = number of viable bacteria after freeze-drying / number of viable bacteria before freeze-drying × 100%. The freeze-drying survival rates of Enterococcus faecium LBEF-01 using different single freeze-drying protective agents are shown in Table 6:

[0068] Table 6 Survival rate of Enterococcus faecium LBEF-01 in different single lyophilization protectants

[0069]

[0070] The results showed that the survival rate of the strains was above 60%, which met the requirements of freeze-dried bacterial powder for the survival rate of the strains. Among them, the survival rate of the strains using 5% oligofructose in the sugar protective agent was the highest, the survival rate of the strains using 2% glycerol in the alcohol protective agent was the highest, and the survival rate of the strains using 15% skim milk powder in the composite protective agent was the highest.

[0071] According to the results in Table 6, oligofructose, glycerol and skimmed milk powder were selected to further determine the effect of the composite freeze-drying protective agent on the freeze-dried survival rate of Enterococcus faecium LBEF-01. The protective agent with a concentration of 5% oligofructose, 2% glycerol and 15% skimmed milk powder was prepared and dissolved in sterile water, and then sterilized at 115℃ for 20 minutes and mixed with Enterococcus faecium LBEF-01 cells. Three parallel groups were set up and the average viable rate was calculated before and after freeze-drying. The results are shown in Table 7:

[0072] Table 7 Survival rate of Enterococcus faecium LBEF-01 in composite freeze-dried protective agent

[0073]

[0074] The results showed that the average freeze-drying survival rate of Enterococcus faecium LBEF-01 using 5% oligofructose, 2% glycerol and 15% skimmed milk powder was 94%-100%.

[0075] Example 7 Determination of fecal bile acid in a canine model fed with Enterococcus faecium LBEF-01

[0076] The results of in vitro identification showed that Enterococcus faecium LBEF-01 has good bile salt tolerance and the characteristics of expressing bile salt hydrolase. In order to further verify the in vivo efficacy, three healthy dogs were selected as the control group, and five dogs with obvious tear stain symptoms were selected to join the experimental group. The control group was fed a complete dog diet (naughty brand, purchased from China Pet Foods Co., Ltd.), and the experimental group was fed lyophilized powder of Enterococcus faecium LBEF-01 on the basis of the complete diet (2g per day, with a viable count of not less than 1×10 9CFU / kg) for one month. Feces were collected once a week to measure the total bile acid content.

[0077] The composition of Enterococcus faecium LBEF-01 freeze-dried powder is as follows (by mass percentage): 1% live Enterococcus faecium LBEF-01 bacteria, 15% skimmed milk powder, 2% glycerol, 5% oligofructose, and 77% sterile water.

[0078] Method for determination of total bile acid content in feces: Feces of dogs in the control group and experimental group were collected once a week and freeze-dried to remove excess water in the feces. 0.1g of dry feces was taken and resuspended in 100ml of anhydrous ethanol (the volume ratio of the two was 1:1000), ice bathed, and vortexed to mix. After centrifugation at 4℃, 12000rpm, and 10min, the supernatant was taken and the total bile acid content was detected and calculated using the fecal TBA content determination kit (biosharp), and the average value of each group was taken and statistically analyzed.

[0079] Table 8 Mean values ​​of total bile acid in feces of canine models fed with Enterococcus faecium LBEF-01

[0080]

[0081] As shown in Table 8 and Figure 6 As shown, the fecal bile acid of dogs with obvious tear stain symptoms before the experiment was lower than that of healthy dogs. When fed with Enterococcus faecium LBEF-01 for one month, the fecal bile acid content of dogs increased significantly, and the total fecal bile acid content was similar to that of the control group.

[0082] Example 8 Blood cholesterol measurement of canine model fed with Enterococcus faecium LBEF-01

[0083] Using the canine model of Example 7, plasma from the control group and the experimental group was collected intravenously at the beginning and end of the experiment, and the supernatant was obtained by centrifugation at 4000 rpm for 10 min. A standard curve was established according to the instructions of the free cholesterol (FC) content detection kit (Beijing Solebow), and the plasma total cholesterol content of the control group and the experimental group was measured.

[0084] Table 9 Mean blood cholesterol of canine model fed with Enterococcus faecium LBEF-01

[0085]

[0086]

[0087] As shown in Table 9 and Figure 7 As shown, the plasma cholesterol content of the experimental group dogs with obvious tear marks before the experiment was significantly higher than that of the control group. After feeding the probiotic Enterococcus faecium LBEF-01 for one month, the average blood cholesterol of the experimental group decreased.

[0088] Example 9 Verification of tear stain removal effect of feeding Enterococcus faecium LBEF-01 to a canine model

[0089] Five canine models with obvious tear stain symptoms from the experimental group of Example 7 were fed with freeze-dried powder of Enterococcus faecium LBEF-01 (2 g per day, with a viable count of not less than 1×10 9 CFU / kg), and observe the dogs' eyes for tears and secretions every day. When no tears or secretions are observed in the dogs' eyes, stop taking the freeze-dried powder, and continue to observe for one week without recurrence of tear marks. It can be considered that the tear mark removal effect of the dogs has improved. The statistical results of the time from enrollment to cure of the experimental dogs are shown in Table 10:

[0090] Table 10 Recovery time of tear stain removal in canine model fed with Enterococcus faecium LBEF-01 lyophilized powder

[0091]

[0092] As shown in Table 10, the average tear recovery time after feeding the freeze-dried powder of Enterococcus faecium LBEF-01 was 44 days, and the average time without ocular secretions was about 23 days. As shown in Figure 8, the ocular secretions and tearing symptoms of dogs were improved after feeding the freeze-dried powder of Enterococcus faecium LBEF-01.

[0093] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered as protections of the present invention.

Claims

1. A strain of canine-derived Enterococcus faecium, named Enterococcus faecium LBEF-01, was deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit date of August 15, 2024 and a deposit number of GDMCCNO.65013.

2. Use of the Enterococcus faecium LBEF-01 described in claim 1 as a live bacterium in a probiotic preparation.

3. The use according to claim 2, wherein the preparation is in the form of a freeze-dried powder preparation or a bacterial suspension preparation, preferably a freeze-dried powder preparation.

4. A probiotic freeze-dried powder preparation, characterized in that: The invention comprises live Enterococcus faecium LBEF-01 bacterial cells and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises one or more of sugars, alcohols and composite protective agents.

5. The probiotic freeze-dried powder preparation according to claim 4, characterized in that: The sugar is selected from at least one of lactose, glucose, trehalose, dextran, xylooligosaccharides or fructo-oligosaccharides, preferably fructo-oligosaccharides; the alcohol is selected from at least one of glycerol, mannitol or sorbitol, preferably glycerol; the composite protective agent is selected from at least one of skimmed milk powder, maltodextrin or soluble starch, preferably skimmed milk powder.

6. The probiotic freeze-dried powder preparation according to claim 4, characterized in that: In terms of weight percentage, it contains live Enterococcus faecium LBEF-01 bacteria, 10%-20% skimmed milk powder, 1.5%-2% glycerol, 3%-5% oligofructose, and the balance is sterile water.

7. Use of the Enterococcus faecium LBEF-01 according to claim 1 or the probiotic freeze-dried powder preparation according to any one of claims 4 to 6 in the preparation of pet products for improving hepatic tear marks caused by abnormal enterohepatic circulation in pets, bacterial diarrhea or related diseases caused by intestinal flora imbalance.

8. The use according to claim 7, characterized in that: The pet products include pet medicines, pet food and pet health products.

9. The method for preparing the probiotic freeze-dried powder preparation according to any one of claims 4 to 6, characterized in that: The method is as follows: The Enterococcus faecium LBEF-01 colony was inoculated into a BHI liquid culture medium or an MRS liquid culture medium, and statically cultured at 30-37° C. for 14 h-16 h to obtain an activated bacterial solution; The activated bacterial liquid is inoculated into BHI liquid medium or MRS liquid medium at a ratio of 1%-2%, and then cultured at 30-37°C. The fermentation liquid is centrifuged to collect the bacterial cells, and the bacterial cells are resuspended and washed with sterile water, and then a freeze-drying protective agent is added; Probiotic freeze-dried powder is obtained after low-temperature freeze-drying.

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