Cat-source enterococcus faecalis ZYpet-013 with cholesterol-lowering activity and application of cat-source enterococcus faecalis ZYpet-013

Abstract

The invention provides cat-source enterococcus faecalis ZYpet-013 with cholesterol lowering activity and application of the cat-source enterococcus faecalis ZYpet-013. The cat-source enterococcus faecalis ZYpet-013 is derived from intestinal tracts of healthy pet cats, and the preservation number of the cat-source enterococcus faecalis ZYpet-013 is CGMCC No. 32009. The enterococcus faecalis ZYpet-013 is used for preparing products for degrading cholesterol, a cat food spraying composition stably loading the strain and a low-temperature spraying process are developed in a matched mode, the cat food with the cholesterol reducing function is prepared, through daily feeding, the levels of serum total cholesterol and triglyceride of pet cats are safely and effectively reduced, blood fat metabolism is improved, and the cholesterol reducing function is achieved. The requirements of industrial production and shelf life stability are met.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of microbial technology and pet nutrition and health, and specifically relates to a feline Enterococcus faecalis ZYpet-013 with cholesterol-lowering activity and its application. Background Technology

[0002] With the rapid increase in the number of pets and the refinement of feeding patterns, the proportion of animal fat in pet cats' daily diets continues to rise. High-energy, high-fat diets easily lead to metabolic diseases such as obesity, hypercholesterolemia, and hyperlipidemia in pet cats, thereby increasing the risk of fatty liver, atherosclerosis, cardiovascular disease, and pancreatitis. Clinical and epidemiological surveys show that some middle-aged and older pet cats have elevated serum total cholesterol (TC) and triglycerides (TG), posing a potential threat to their health and lifespan.

[0003] Currently, interventions for abnormal cholesterol levels mainly fall into two categories: drug therapy and nutritional intervention. Drug therapy, such as statins, is widely used in humans, but its long-term safety, tolerability, and adherence to use in cats are highly uncertain, making it unsuitable as a routine nutritional management method; furthermore, it requires monitoring of liver and kidney function, resulting in high costs. Nutritional interventions commonly involve reducing dietary fat levels, adding soluble dietary fiber, and phytosterols, which can improve blood lipids to some extent, but suffer from limited cholesterol-lowering effects, slow onset of action, and significant individual variability.

[0004] In recent years, the role of probiotics in regulating cholesterol metabolism has attracted increasing attention. Existing studies have reported that some lactic acid bacteria from human or fermented foods possess bile salt hydrolase (BSH) activity, which can promote the conversion of cholesterol into bile acids through the deconjugation of bile salts. Simultaneously, lactic acid bacteria can remove cholesterol from the intestinal lumen through cell surface adsorption and co-precipitation, thereby lowering serum cholesterol levels. However, currently available probiotics for pets primarily focus on diarrhea prevention, gut health, and immune regulation, with relatively few systematic studies on pet cholesterol metabolism and products demonstrating a clear "cholesterol-lowering effect." Furthermore, existing probiotics are mostly derived from the human gut, dairy products, or fermented plant foods, which differ from the gut microbiota of cats. Their adaptability and colonization ability in the cat's gut are limited, and the stability and efficacy of long-term use are difficult to guarantee.

[0005] Furthermore, in the pet food industry, to ensure the process stability and sensory quality of extruded cat food, it often needs to undergo high-temperature and high-pressure processing, which can significantly reduce the activity of probiotics. Therefore, even when probiotic powder is added to dry pet food, it is often difficult to maintain a sufficient number of live bacteria in the final product due to processing limitations.

[0006] In summary, there is an urgent need for a proprietary lactic acid bacteria strain and its application scheme that originates from the cat's own intestines, has clear cholesterol-lowering activity, is adapted to the cat's gastrointestinal environment, and can be stably loaded onto the surface of cat food by spraying, in order to achieve safe, effective, and industrially feasible nutritional regulation of cholesterol levels in pet cats.

[0007] To address the problems existing in the prior art, the present invention aims to provide a feline Enterococcus faecalis ZYpet-013 with cholesterol-lowering activity and its application, and to develop a cat food spraying composition and low-temperature spraying process that stably loads this strain, so as to prepare a cholesterol-lowering functional cat food. Through daily feeding, it can safely and effectively reduce the serum total cholesterol and triglyceride levels of pet cats, improve lipid metabolism, and is suitable for industrial production and shelf-life stability requirements.

[0008] The objective of this invention is achieved through the following technical solution: The first aspect of the present invention provides a feline Enterococcus faecalis ZYpet-013 with cholesterol-lowering activity, which was deposited on September 20, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32009.

[0009] Furthermore, the Enterococcus faecalis ZYpet-013 has the 16S rRNA gene sequence shown in SEQ ID No:1:

[0010] A second aspect of the present invention provides the application of the aforementioned feline Enterococcus faecalis ZYpet-013 in the preparation of cholesterol-degrading products.

[0011] Furthermore, the products include pet food.

[0012] The third aspect of the present invention provides the application of the feline Enterococcus faecalis ZYpet-013 in the preparation of an antibacterial agent, wherein the antibacterial agent can inhibit Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Bacillus subtilis and / or Shigella dysenteriae.

[0013] A fourth aspect of the present invention provides a pet food spray composition capable of degrading cholesterol, comprising an active ingredient for degrading cholesterol and a food-grade carrier, wherein the active ingredient comprises the aforementioned feline Enterococcus faecalis ZYpet-013.

[0014] Furthermore, the carrier is one or more of maltodextrin, inulin, fructooligosaccharides, oligofructose, and whole milk powder.

[0015] Furthermore, the content of feline Enterococcus faecalis ZYpet-013 in the composition is [missing information]. CFU / g or CFU / mL, preferably CFU / g or CFU / mL.

[0016] The fifth aspect of this invention provides a cat food with cholesterol-lowering effects, using conventional cat food as a base, and spraying the surface of it with the pet food coating composition described in the fourth aspect, such that the content of feline Enterococcus faecalis ZYpet-013 in the resulting cat food is [missing information]. CFU / g or CFU / mL, preferably CFU / g or CFU / mL.

[0017] Furthermore, the preparation of this cat food employs a two-stage spraying process: first, an oil is sprayed, wherein the oil is one or more of chicken oil, fish oil, and vegetable oil; second, the pet food spraying composition described in the fourth aspect is sprayed.

[0018] Preferably, the cat food base is commercially available dry cat food prepared through extrusion or baking processes. Preferably, after the spray-coated cat food is stored at room temperature and away from light for 6–12 months, the viable count of the feline Enterococcus faecalis ZYpet-013 remains no less than [a certain percentage]. CFU / g.

[0019] Information on strain preservation: Enterococcus faecalis ZYpet-013, deposited at: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Accession number: CGMCCNo.32009; Deposit date: September 20, 2024; Classification and nomenclature: Enterococcus faecalis.

[0020] The advantages of this invention compared to the prior art are as follows: 1. This invention screened and obtained a strain of Enterococcus faecalis ZYpet-013 with a clear source and strong targeting. It is derived from the intestine of a healthy pet cat and is more adapted to the cat's gastrointestinal environment than human or food-derived lactic acid bacteria. It has better potential colonization and symbiotic ability and can specifically regulate cat cholesterol metabolism, thereby improving the safety and effectiveness of the application. At the same time, this strain has significant cholesterol-lowering activity in vitro. In a cholesterol model system containing bile salts, the cholesterol removal rate can reach 50-63% after 48 hours, and it also has strong bile salt hydrolase activity. 2. The Enterococcus faecalis ZYpet-013 described in this invention has good acid and bile salt tolerance and is suitable for oral administration: Enterococcus faecalis ZYpet-013 has a high survival rate in simulated cat gastric juice and bile salt environment, and can pass smoothly through the stomach after oral administration, reaching the small intestine and colon, ensuring that it plays a continuous role in microecological regulation and cholesterol metabolism improvement in vivo. 3. The present invention further prepares a cat food spraying composition containing the aforementioned Enterococcus faecalis ZYpet-013, and uses a two-stage low-temperature spraying process to avoid high-temperature processing from destroying the activity of the strain, so that the strain is stably attached to the surface of the cat food in the form of live bacteria, ensuring the number of live bacteria and functional effectiveness of the end product. Moreover, this process can be directly implemented on existing cat food factory equipment, and has good industrial feasibility and economy. 3. This invention provides a cholesterol-lowering functional cat food with a spray-coated finish, exhibiting excellent shelf-life stability: the spray-coated cat food maintains a high number of viable bacteria under normal temperature storage conditions and can maintain this level of activity within a shelf life of 6–12 months. A bacterial count level of CFU / g or higher ensures that the product has substantial functional effects throughout its entire life cycle; 4. Animal experiments have confirmed that the cholesterol-lowering effect is significant: In obese pet cats, continuous feeding of cat food sprayed with the Enterococcus faecalis ZYpet-013 composition for 8 weeks can significantly improve the serum total cholesterol and triglyceride levels of obese cats, proving that the cat food of the present invention has a clear cholesterol-lowering function. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The phylogenetic tree of Enterococcus faecalis ZYpet-013 is shown; Figure 2 The presence of Enterococcus faecalis ZYpet-013 in The activity of bile salt hydrolase on agar plates; Figure 3 It is the lyophilized form of Enterococcus faecalis ZYpet-013 bacterial powder. Detailed Implementation

[0022] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0025] The MRS liquid culture medium described in the following examples consists of: 10 g peptone, 10 g beef extract, 10 g yeast extract, 20 g glucose, 5 g sodium acetate, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, and 0.25 g manganese sulfate. These are dissolved in deionized water, brought to a final volume of 1 L, and the pH is adjusted to 6.0. The medium is then autoclaved at 121°C for 15 min. The MRS solid culture medium is prepared by adding 17 g agar per L to the liquid culture medium.

[0026] Example 1: Isolation and identification of feline Enterococcus faecalis ZYpet-013 1. Isolation and purification of strains (1) Isolation of strains: Fresh fecal samples from healthy cats were collected from cat shelters, pet hospitals, and stray cat rescue stations in Luohe City, Henan Province. 1 g of fecal sample was weighed and placed in a 50 mL sterile centrifuge tube, and 9 mL of sterile water was added. The sample was thoroughly shaken to disperse it evenly, serving as a 10-fold initial dilution. This suspension was used as the starting solution for continuous 10-fold serial dilutions until… 10 times, respectively, take 100 μL 10 times, Doubled The diluted solution was spread evenly on MRS solid medium plates and incubated in an anaerobic environment at 37°C for 48 h to obtain candidate colonies of lactic acid bacteria.

[0027] (2) Purification of the strain: Select milky white raised colonies with different morphology and size from the above plates and inoculate them onto new MRS solid plates for isolation and purification using the three-zone streak method; after streaking, the plates are incubated at 37°C under anaerobic conditions for 48 h. Repeat the above streak culture steps for a total of 3 rounds of subculturing until a pure culture strain with a single morphology is obtained.

[0028] (3) Morphological identification: Gram staining was used to identify the morphological characteristics of lactic acid bacteria. The specific operation was as follows: 1 drop of sterile water was added to a clean glass slide, a small amount of single colony was picked up and evenly spread on the surface of the glass slide, and it was gently shaken and air-dried over an alcohol lamp flame; then, ammonium oxalate crystal violet staining solution was added for about 1 min, excess staining solution was gently rinsed with sterile water and air-dried again; iodine solution was added for mordanting for about 1 min, washed with water and air-dried, then decolorized with 95% ethanol for about 20 s, quickly rinsed with sterile water and dried; then safranin staining solution was added for 1 min, washed with water and dried, and the morphology of the bacteria and the results of the Gram reaction were observed under an optical microscope.

[0029] (4) Catalase experiment: Add 100 μL of 3% catalase to a sterile empty culture dish. The solution was then used to pick a single colony of the test strain with an inoculation loop and gently touched... Observe the surface of the droplet to see if bubbles are generated immediately. If obvious bubbles are observed, the strain is determined to be catalase positive; if no bubbles are observed, it is determined to be catalase negative.

[0030] (5) Glucose gas production experiment: First, a single colony of lactic acid bacteria was inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain an activated bacterial suspension. The Durham tube was inverted and placed into a test tube containing MRS liquid medium. The aforementioned bacterial suspension was added at an inoculation rate of 1% (v / v). The tube was then incubated at 37℃ for 7 days. The presence of bubbles in the Durham tube was observed. If obvious bubbles appeared in the Durham tube, it indicated that the strain fermented glucose and produced gas, and was a heterofermentative lactic acid bacteria. If no bubbles were observed in the Durham tube, it was a homofermentative lactic acid bacteria.

[0031]

[0032] As shown in Table 1, strain ZYpet-013 is milky white, round, slightly convex, and has neat edges. It is a homofermentative lactic acid bacteria, and its Gram staining result is positive, while its catalase result is negative.

[0033] 2. Strain identification The strain was anaerobically cultured on MRS plates at 37℃ for 48 h, and single colonies were picked for PCR. Primers used were the universal 16S rRNA primers 27F and 1492R. PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 90 s, 35 cycles; final extension at 72℃ for 5 min. The obtained 16S rRNA was compared with sequences of known strains from GenBank using BLAST analysis, and a phylogenetic tree was constructed using MEGA-X software (see [link to BLAST analysis]). Figure 1 Phylogenetic analysis identified strain ZYpet-013 as Enterococcus faecalis and named it Enterococcus faecalis ZYpet-013.

[0034] The specific sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No: 2); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No: 3).

[0035] The Enterococcus faecalis ZYpet-013 has a 16S rRNA gene sequence as shown in SEQ ID No:1.

[0036] Example 2: Experiment on the hydrophobicity and self-aggregation of Enterococcus faecalis ZYpet-013 cells To evaluate the cell surface hydrophobicity and self-aggregation ability of the Enterococcus faecalis ZYpet-013 strain isolated in Example 1, the following method was used in this example: First, single colonies of the above-mentioned lactic acid bacteria were picked and inoculated into test tubes containing 20 mL of MRS liquid medium. The cultures were then anaerobic at 37°C for approximately 16 h to allow the bacteria to grow to the late logarithmic or stationary phase. After incubation, the cultures were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the bacteria were washed twice with PBS buffer. The bacterial pellet was then resuspended in PBS, and the absorbance at 600 nm was measured and recorded as λ. , as the initial bacterial concentration.

[0037] To determine the hydrophobicity of the cell surface, 3 mL of the bacterial suspension was added to a test tube containing 1 mL of xylene. The mixture was vortexed thoroughly for approximately 2 minutes to form a two-phase mixture. The mixture was then incubated at 37°C for 30 minutes until the organic and aqueous phases completely separated. The aqueous phase was carefully aspirated, and its absorbance at 600 nm was measured and recorded as _____. .

[0038] Cell surface hydrophobicity (%) is calculated using the following formula:

[0039] For the self-aggregation assay, 3 mL of the treated bacterial suspension was placed at 37°C and allowed to stand. The supernatant was collected at 0 h and 8 h, and the absorbance at 600 nm was measured. This absorbance was recorded as follows: .

[0040] Self-aggregation (%) is calculated using the following formula:

[0041] The hydrophobicity and self-aggregation experiments of the strains were all performed in triplicate.

[0042]

[0043] As shown in Table 2, Enterococcus faecalis ZYpet-013 has a hydrophobicity of 37.9% and a self-aggregation rate of 58.7%, indicating that the strain has a strong ability to colonize the intestines.

[0044] Example 3: Physiological and biochemical characteristics of feline Enterococcus faecalis ZYpet-013 This embodiment evaluates the physiological and biochemical characteristics of Enterococcus faecalis ZYpet-013, specifically including: Enterococcus faecalis ZYpet-013 was inoculated into MRS liquid medium and cultured at 37°C. The bacterial suspension was then adjusted to OD600 = 0.8, and subsequently inoculated into MRS media with different treatments in proportion. The acid-base tolerance, salt tolerance, and temperature tolerance of the strain were evaluated by adjusting the pH of the medium (3.0 to 10.0), the NaCl concentration (3.0% and 6.5%), and the culture temperature (5°C to 50°C).

[0045] After incubation under appropriate conditions, the growth of the strain was assessed by absorbance at 600 nm, and confirmed by visual observation of turbidity. Uninoculated MRS medium served as a control. Based on the measured OD600 values, the growth of the strain was categorized into no growth (≤0.2), weak growth (0.2 to 0.6), and significant growth (>0.6), thereby determining the tolerance of Enterococcus faecalis ZYpet-013 under various experimental conditions.

[0046]

[0047] As shown in Table 3, Enterococcus faecalis ZYpet-013 does not grow at 5°C, but shows significant growth at 10°C, 45°C, and 50°C, indicating good high-temperature tolerance but not suitability for low-temperature environments. It can grow normally in media containing 3.0% and 6.5% NaCl, showing strong salt tolerance. It grows weakly at pH 3.0, but shows significant growth in the pH range of 3.5 to 10.0, indicating that this strain can adapt to a wide pH range from slightly acidic to alkaline, and also has a certain tolerance to strongly acidic conditions.

[0048] Example 4: Test results of bile salt hydrolase in feline Enterococcus faecalis ZYpet-013 This embodiment evaluates the bile salt hydrolysis activity of Enterococcus faecalis ZYpet-013, specifically including: The experiment used a modified MRS solid culture medium, supplemented with 0.5% sodium glycocholate (GDCA) and 0.037% calcium chloride (CCl4) to the basic formulation. To enhance the visualization of the precipitate formed by the deconjugated bile acids and calcium ions produced during hydrolysis, strain ZYpet-013 was streaked onto the aforementioned plates and anaerobically cultured at 37°C for 48 h. The presence of a typical milky-white precipitate ring around the colony was used as a screening criterion. This precipitate ring was approximately 2 mm wide, with blurred edges, irregular shape, and a misty appearance surrounding the colony, exhibiting a clear contrast to the transparent background of the culture medium.

[0049] The strain was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24 h. 10 mL of the culture was collected by centrifugation at 8000 rpm for 10 min at 4°C. The cells were resuspended in 0.1 M phosphate buffer (pH 7.0) and sonicated to obtain a free cell extract. A certain volume of the extract was added to a reaction system containing sodium glycocholate (GDCA, final concentration 10 mM). The reaction was incubated at 37°C for 30 min, and then 15% trichloroacetic acid was added to terminate the reaction. The supernatant was collected by centrifugation, and the concentration of released bile acids was determined using the ninhydrin colorimetric assay. The hydrolysis rate was calculated to characterize the bile salt hydrolysis activity of the strain. The experiment was performed in triplicate with a blank control to eliminate non-enzymatic background.

[0050] The results are as follows Figure 2 As shown, Enterococcus faecalis ZYpet-013 forms a distinct milky-white precipitate band around its colonies, exhibiting a misty, ring-like characteristic that contrasts sharply with the transparent background of the culture medium itself. This precipitate ring is approximately 2 mm wide, with somewhat blurred edges, and is irregularly distributed around the colonies.

[0051]

[0052] Table 4 shows that the bile salt hydrolysis rate of Enterococcus faecalis ZYpet-013 was 86.14%, indicating that this strain has high bile salt hydrolysis activity.

[0053] Example 5: Evaluation of cholesterol degradation activity of feline Enterococcus faecalis ZYpet-013 This embodiment evaluated the cholesterol degradation activity of Enterococcus faecalis ZYpet-013, specifically including: The strain was inoculated into MRS liquid medium containing 100 µg / mL water-soluble cholesterol. The cholesterol was first dissolved in anhydrous ethanol and emulsified with 0.3% (v / v) Tween 80. After anaerobic incubation at 37°C for 48 h, the medium was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to determine the residual cholesterol content. For the determination, the supernatant was first treated with trichloroacetic acid, and then sulfuric acid-glacial acetic acid-ferric (SAF-Fe) solution was added. The colorimetric reagent was used to measure the absorbance at a wavelength of 560 nm using a spectrophotometer, and the cholesterol concentration was calculated using a standard curve, thereby converting the cholesterol removal rate of Enterococcus faecalis ZYpet-013.

[0054]

[0055] The results are shown in Table 5. The cholesterol removal rate of Enterococcus faecalis ZYpet-013 was 62.86%, indicating that this strain has a strong cholesterol removal ability in vitro.

[0056] Example 6: Simulated artificial gastrointestinal fluid experiment of feline Enterococcus faecalis ZYpet-013 To evaluate the survival ability of Enterococcus faecalis ZYpet-013 isolated in Example 1 in the gastrointestinal environment, a tolerance test was conducted using in vitro simulated gastric and intestinal fluids, as detailed below: (1) Preparation of simulated gastric juice: Weigh 0.35 g of pepsin and dissolve it in 0.2% sterile physiological saline. After mixing thoroughly, adjust the pH of the solution to 2.0 with hydrochloric acid, and then add 0.2% sterile physiological saline to make the total volume 100 mL. The resulting solution is filtered through a 0.22 μm microporous membrane for sterilization and is used as simulated gastric juice for later use.

[0057] (2) Preparation of simulated intestinal fluid: Weigh 0.1 g trypsin, 1.8 g bovine bile salt and 1.1 g sodium bicarbonate, add them to 0.2% sterile physiological saline to dissolve, adjust the pH of the solution to 6.8, and make up to 100 mL with 0.2% sterile physiological saline. Filter the solution through a 0.22 μm microporous membrane to remove bacteria. The resulting solution is used as simulated intestinal fluid.

[0058] (3) Bacterial suspension treatment and counting: The bacterial suspension was activated with MRS liquid medium and the bacterial concentration was approximately A CFU / mL suspension of Enterococcus faecalis ZYpet-013 was added to 20 mL of simulated gastric fluid at a 2% (v / v) inoculation rate and incubated for 3 h under suitable conditions. Samples were taken at 0 h and 3 h after treatment, and viable counts were performed on MRS agar plates using the serial dilution-spreading method to obtain the viable bacterial counts before and after simulated gastric fluid treatment. Subsequently, 100 μL of the bacterial suspension from the 3 h incubation in gastric fluid was added to 900 μL of simulated intestinal fluid, and incubation was continued for 4 h under the set conditions. Samples were also taken at 0 h and 4 h after intestinal fluid treatment, and viable bacterial counts were performed on MRS agar plates using the dilution-spreading method. The survival rate of the strain in simulated gastric and intestinal fluids was calculated based on these counts. All experiments were performed in triplicate to ensure data reliability.

[0059]

[0060] The results are shown in Table 6. Enterococcus faecalis ZYpet-013 showed good survival ability in simulated gastrointestinal fluid: its concentration in gastric fluid decreased from 9.00 CFU / mL at 0 h to 7.76 CFU / mL at 3 h, and in intestinal fluid it decreased from 6.76 CFU / mL at 0 h to 5.99 CFU / mL at 4 h. This indicates that the strain can maintain a high survival level under stress conditions such as gastric acid and bile salts, and has a certain potential ability to reach the intestine orally.

[0061] Example 7: Broad-spectrum antibacterial activity of feline Enterococcus faecalis ZYpet-013 The broad-spectrum antibacterial activity against Enterococcus faecalis ZYpet-013 was evaluated using a modified agar well diffusion method, specifically including: Pseudomonas aeruginosa (CICC 23694) was selected. T Staphylococcus aureus (ATCC 29213) T Listeria monocytogenes (CICC23929) T Escherichia coli (CICC 24189) T Bacillus subtilis (CICC 10275) T ) and Shigella dysenteriae (CICC 23829) T As indicator strains, each pathogen was cultured in LB liquid medium at 37°C and 180 rpm until the logarithmic growth phase (approximately...). (CFU / mL), take 200 μL of bacterial suspension and spread it evenly on LB agar plates. After surface absorption, use a sterile punch to make 10 mm diameter holes, and add 200 μL of Enterococcus faecalis ZYpet-013 fermentation broth (approximately CFU / mL) to each hole. (CFU / mL, obtained by incubation at 37°C for 16 h). Uninoculated MRS medium served as a negative control, and penicillin (10 μg / mL) served as a positive control. After pre-diffusion at 37°C for 2 h, plates were incubated upside down for 24 h, and the diameter of the inhibition zone was observed and measured to evaluate the broad-spectrum antibacterial activity of Enterococcus faecalis ZYpet-013 against the above-mentioned pathogens.

[0062] The results are shown in Table 7. Enterococcus faecalis ZYpet-013 exhibited strong in vitro antibacterial activity against a variety of pathogenic bacteria. Its inhibition zone rating against Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus was "++", with corresponding inhibition zone diameters of approximately 14.00–18.00 mm; its inhibitory effect against Listeria monocytogenes and Escherichia coli was "+++", with inhibition zone diameters of approximately 18.00–22.00 mm; and its antibacterial effect against Shigella dysenteriae was even stronger, reaching "++++", demonstrating the most significant antibacterial effect.

[0063] Overall results indicate that Enterococcus faecalis ZYpet-013 has good broad-spectrum antibacterial activity.

[0064]

[0065] Example 8: Enterococcus faecalis ZYpet-013 spray composition and its application in commercially available cat food This embodiment provides a spray composition for Enterococcus faecalis ZYpet-013, the preparation method of which is as follows: (1) Preparation of freeze-dried ZYpet-013 bacterial powder: Enterococcus faecalis ZYpet-013 from cats was inoculated into a 3 L MRS fermenter and anaerobically cultured at 37℃ for about 18 h. The bacterial count at the end of fermentation was approximately CFU / mL. The fermentation broth was centrifuged to collect the bacterial cells. Approximately 10% of a food-grade carrier mixture (inulin and fructooligosaccharides) was added based on the wet weight of the bacterial cells. After mixing, the mixture was frozen and lyophilized to obtain ZYpet-013 lyophilized bacterial powder with a viable cell count of approximately [missing information]. CFU / g, at Within the CFU / g range, lyophilized powder such as Figure 3 As shown.

[0066] (2) Preparation of cholesterol-lowering spray composition: The above-mentioned ZYpet-013 freeze-dried bacterial powder and whole goat milk powder were mixed in a certain proportion to prepare a cat-derived Enterococcus faecalis spray composition, so that the content of ZYpet-013 in the composition was controlled at approximately CFU / g.

[0067] (3) Take 100 kg of commercially available dry cat food prepared by extrusion puffing as the base material. First spraying: Slowly roll the cat food base material in a rotary drum spraying device, and spray chicken oil at 1% of the base material mass to evenly cover the surface of the cat food. Second spraying: Continue to roll the cat food base material, and under the condition that the material temperature does not exceed 45℃, spray the ZYpet-013 spraying composition prepared in step (2) at 1% of the base material mass to evenly adhere it to the oiled cat food surface. After spraying, let it cool naturally to room temperature to obtain cat food with cholesterol-lowering effect. Sampling and testing showed that the initial viable count of ZYpet-013 in the obtained cat food with cholesterol-lowering effect was approximately CFU / g.

[0068] (4) Storage stability: The cat food with cholesterol-lowering effects prepared above was stored at room temperature and away from light for 6–12 months, and the viable bacteria count was tested periodically. The results showed that after 12 months of storage, the viable bacteria count of ZYpet-013 in the cat food was still not lower than [a certain value]. CFU / g, preferably maintained at The presence of CFU / g or higher indicates that the two-stage spraying process and carrier system of this invention can maintain a high level of viable bacteria during the shelf life, ensuring the cholesterol-lowering effect of the cat food.

[0069] Example 9: Effect of Enterococcus faecalis ZYpet-013 inoculum on cholesterol levels in mice induced by a high-fat diet To investigate the effect of Enterococcus faecalis ZYpet-013 on cholesterol levels in mice, the following animal experiments were conducted in this example: (1) Experimental animals and grouping: Twenty-four SPF-grade male C57BL / 6J mice, 6 weeks old and weighing (20.0±2.0) g, were selected. The mice were acclimatized in a barrier environment for 7 days before the experiment. The rearing conditions were (22±2)℃, (50±10)% relative humidity, and a light / dark cycle of 12 h / 12 ​​h. They had free access to standard feed and water. All experimental procedures complied with the ethical guidelines for animal experiments and were approved by the animal ethics committee of the institution (Aw42405202-1-10).

[0070] After the adaptation period, the mice were randomly divided into 3 groups of 8 mice each, according to their body weight: Normal control group (Control): fed with basal diet and gavage with 0.2 mL of physiological saline; High-fat model group (Model): fed a high-fat diet and administered 0.2 mL of physiological saline by gavage; Probiotic group: Feed high-fat diet, and administer 0.2 mL of Enterococcus faecalis ZYpet-013 probiotic agent (containing live bacteria count) by gavage. CFU).

[0071] The basal diet consists of ordinary pelleted feed; the high-fat diet increases the fat content to approximately 1.5 to 2 times that of the basal diet.

[0072] (2) Intervention methods: The modeling phase lasted for 8 weeks. During this period, the high-fat model group and the probiotic group were continuously fed a high-fat diet to induce dyslipidemia, while the normal control group was fed a basal diet throughout. Subsequently, a 12-week intervention phase was initiated. Each group continued the original diet plan and was administered the above treatment once daily by gavage, with an administration volume of 0.2 mL / mouse. During the intervention period, the body weight and food intake of each group of mice were recorded regularly, and their general mental state was observed.

[0073] (3) Detection of blood lipid indicators: At the end of the intervention (week 20), blood was collected from the orbital cavity or heart after fasting for 12 hours. After standing and coagulation, the serum was separated by centrifugation. The levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum were detected using a fully automated biochemical analyzer and matching reagent kit. Each sample was tested with duplicate wells. The concentration of each blood lipid indicator was calculated according to the standard curve to evaluate the effect of Enterococcus faecalis ZYpet-013 on improving cholesterol elevation and blood lipid disorders induced by a high-fat diet in mice.

[0074]

[0075] The results are shown in Table 8. Compared with the control group, the serum TC, TG and LDL-C levels in the high-fat model group were significantly increased, while HDL-C was significantly decreased (p<0.05). The absolute values ​​of TC, TG and LDL-C increased by about 3.3 mmol / L, 1.2 mmol / L and 0.5 mmol / L respectively compared with the control group, while HDL-C decreased by about 40%, indicating that the high-fat diet successfully induced significant hypercholesterolemia and dyslipidemia.

[0076] After intervention with Enterococcus faecalis ZYpet-013, the lipid profile of mice was significantly improved: the levels of TC, TG, and LDL-C in the ZYpet-013 group were significantly lower than those in the model group, with reductions of approximately 17%, 50%, and 40%, respectively, while HDL-C was approximately 25% higher than that in the model group (p < 0.05). There were no significant differences in TG and HDL-C between the group and the control group, indicating that ZYpet-013 not only inhibited the increase in cholesterol and atherosclerosis-related indicators caused by a high-fat diet, but also restored "good cholesterol" levels to some extent, thus having an overall effect of improving lipid metabolism and optimizing cholesterol distribution.

[0077] Example 10: Effect of Enterococcus faecalis ZYpet-013 inoculant sprayed on cat food on serum cholesterol levels in obese cats To investigate the effect of the cholesterol-lowering cat food prepared in Example 8 on serum cholesterol levels in obese cats, the following animal experiments were conducted in this example: (1) Experimental animals and grouping: Twelve adult domestic cats aged 15 to 18 months with good physical condition and a body condition score (BCS, 9-point scale) ≥7 were selected and housed individually. All cats had completed routine immunization and deworming before the experiment and had not used antibiotics or other drugs that may alter the gut microbiota for at least one month prior to the experiment.

[0078] After being evenly divided by weight and BCS, the animals were randomly divided into two groups of six each, as follows: Control group: fed commercially available baked dry cat food without spray coating; Probiotics group: Feed the same commercially available cat food with sprayed cat-derived Enterococcus faecalis ZYpet-013.

[0079] The nutritional levels of the basal diets of both groups of cats met the AAFCO (2023) and NRC (2006) cat nutrition standards.

[0080] (2) Feeding conditions and intervention plan: The experimental environment adopted a 12-hour diurnal cycle, with the temperature controlled at 18-26 ℃ and the relative humidity at 40%-70%. The cat houses were kept hygienic by cleaning feces twice a day and disinfecting once a day. The adaptation period was 1 week, during which both groups were fed unsprayed commercially available baked dry cat food. Then, a formal intervention trial lasting 56 days began: the control group continued to be fed unsprayed commercially available cat food; the probiotic group was fed the same commercially available cat food sprayed with feline Enterococcus faecalis ZYpet-013. After spraying, the number of live ZYpet-013 bacteria in the feed was approximately CFU / g. Both groups of cats were fed twice daily with a fixed amount of approximately 35 g per cat each time, and had free access to water.

[0081] (3) Venous blood was collected via hind limb veins before the start of the intervention (day 0) and at the end of the intervention (day 56) after a 12-hour fast at night, and serum was separated. Serum total cholesterol (TC) and triglyceride (TG) levels were measured using an automated biochemical analyzer to evaluate the improvement effect of Enterococcus faecalis-sprayed cat food on existing cholesterol elevation in obese cats.

[0082]

[0083] As shown in Table 9, at baseline (day 0), serum TC and TG levels in the two groups of obese cats were similar, with no significant difference (p > 0.05). After 56 days of intervention, both groups showed a decrease in TC and TG compared to baseline. In the control group, TC and TG decreased by approximately 14% and 28%, respectively, while the ZYpet-013 group showed a greater decrease, with TC decreasing by approximately 22% and TG decreasing by approximately 59%. Compared with the control group at the same time point, the ZYpet-013 group showed a further decrease in TC of approximately 12% and a decrease in TG of approximately 37% on day 56. The results indicate that continuous feeding of Enterococcus faecalis-sprayed cat food can significantly improve serum total cholesterol and triglyceride levels in obese cats on the basis of routine dietary management.

[0084] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A feline Enterococcus faecalis ZYpet-013 strain with cholesterol-lowering activity, characterized in that, It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 20, 2024, with accession number CGMCC No. 32009.

2. The feline Enterococcus faecalis ZYpet-013 according to claim 1, characterized in that, The Enterococcus faecalis ZYpet-013 has a 16S rRNA gene sequence as shown in SEQ ID No:

1.

3. The application of the feline Enterococcus faecalis ZYpet-013 as described in claim 1 or 2 in the preparation of cholesterol-degrading products.

4. The application according to claim 3, characterized in that, The products include pet food.

5. The application of *Enterococcus faecalis* ZYpet-013 from felines as described in claim 1 or 2 in the preparation of antibacterial agents, characterized in that... The antibacterial agent can inhibit Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Bacillus subtilis and / or Shigella dysenteriae.

6. A pet food spray composition capable of degrading cholesterol, characterized in that, It includes an active ingredient and a carrier that degrades cholesterol, said active ingredient including the feline Enterococcus faecalis ZYpet-013 as described in claim 1 or 2.

7. The pet food spraying composition according to claim 6, characterized in that, The carrier is one or more of maltodextrin, inulin, fructooligosaccharides, and whole milk powder.

8. The pet food spraying composition according to claim 6, characterized in that, The content of feline Enterococcus faecalis ZYpet-013 in the composition is: CFU / g or CFU / mL.

9. A cat food with cholesterol-lowering effects, characterized in that, Using conventional cat food as a base, a pet food coating composition as described in any one of claims 6 to 8 is sprayed onto its surface, such that the content of feline Enterococcus faecalis ZYpet-013 in the resulting cat food is [missing information]. CFU / g or CFU / mL.

10. The cat food with cholesterol-lowering effect according to claim 9, characterized in that, The cat food is prepared using a two-stage spraying process: first, oil is sprayed on, and then the pet food spraying composition as described in any one of claims 6 to 8 is sprayed on.

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