Enterococcus hirae, curcuma composition and application thereof in pet weight management

Abstract

The application provides an Enterococcus hirae and curcuma composition and application thereof in pet weight management, and the composition comprises: (a) Enterococcus hirae ZYpet-011, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 32007, and the preservation date is September 20, 2024; (b) curcuma and / or curcuma extract. The Enterococcus hirae is compounded with curcuma for the first time, and through the synergistic effect of the strain and the plant ingredients, the safe, mild and long-acting improvement of obese pets' dyslipidemia, liver burden and chronic inflammation is realized, the synergistic lipid-lowering, anti-inflammatory and liver protection are realized, the composition effect is significantly better than that of a single component, a long-term feeding, stable effect weight and metabolism management solution is provided for obese pets, and the shortage of the prior art is made up.

Description

Technical Field

[0001] This invention belongs to the field of pet microbial products technology, and specifically relates to a combination of Enterococcus haematobacterium and turmeric and its application in pet weight management. Background Technology

[0002] In recent years, with the rapid growth in pet ownership and the popularization of refined pet care, obesity in pet cats has become increasingly prominent, with a high proportion of overweight and obese adult cats kept indoors in cities. Obesity not only leads to excessive accumulation of weight and body fat in cats, but is also often accompanied by metabolic disorders such as dyslipidemia, insulin resistance, fatty liver, and chronic low-grade inflammation, severely reducing the healthy lifespan and quality of life of pets. How to manage the weight of obese cats safely and sustainably has become an urgent need for veterinary clinics and the pet food industry.

[0003] Current intervention methods for obese cats mainly include using prescription diets with restricted energy intake, increasing exercise, and adding dietary fiber or L-carnitine to the diet. While these methods can reduce weight to some extent, they generally suffer from poor compliance, rapid weight loss which can induce fatty liver disease, and stress or decreased appetite in some cats. They fail to achieve gentle, safe, and long-term sustainable weight management results and cannot meet the market demand for high-quality, scientifically sound weight loss solutions.

[0004] The gut microbiota is closely related to metabolic diseases such as obesity. Some probiotics, such as lactic acid bacteria and enterococci, can improve lipid metabolism through bile salt hydrolysis, cholesterol reduction, short-chain fatty acid production, and inflammation regulation. Currently, the number of probiotic products for pets is gradually increasing, but most strains originate from human gut microbiota or dairy fermentation systems. Their colonization ability in the cat gut, long-term stability, and systemic intervention effects on obesity-related metabolic indicators remain unclear. Feline strains are more adapted to the feline gut environment and are more likely to colonize stably and exert their effects in cats. However, existing technologies related to feline strains mainly focus on improving diarrhea or general gut health, lacking systematic research and product solutions specifically for obese or dyslipidemia-prone cats.

[0005] Plant-derived active ingredients have garnered significant attention for their role in the nutritional regulation of metabolic diseases. Turmeric (Curcuma longa) and its main active ingredient, curcumin, possess antioxidant, anti-inflammatory, and lipid metabolism-regulating activities, demonstrating potential in various animal models and human studies to improve blood lipids and reduce hepatic steatosis. While some existing pet foods or health supplements contain turmeric powder or extract, these are primarily used for general antioxidant or health maintenance claims, lacking appropriate dosage and formulation design for obese cats, and lacking systematic efficacy verification in obese cats.

[0006] In summary, existing nutritional intervention methods for obese cats have significant limitations: they rely on energy restriction, conventional dietary fiber, or human probiotics, lacking specialized strains adapted to the feline gut environment that possess both colonization ability and lipid metabolism regulation function; the application of plant active ingredients such as turmeric in the pet industry is rudimentary, and a systematic intervention program for metabolic abnormalities in obese cats has not been formed; at the same time, there is a lack of mature technologies for the compatibility of strains and plant active ingredients to synergistically regulate lipid metabolism and inflammation, making it difficult to achieve safe, long-term, and fundamental weight and metabolic management in obese cats. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to provide a combination of Enterococcus helicobacter and turmeric and its application in pet weight management. Through the synergistic effect of the bacterial strain and plant components, it achieves safe, gentle, and long-lasting improvement of dyslipidemia, liver burden, and chronic inflammation in obese pets, providing a long-term, stable weight and metabolic management solution for obese pets, thus overcoming the shortcomings of the prior art.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The first aspect of this invention provides a composition of Enterococcus haematobacterium and turmeric for pet weight management, the composition comprising:

[0010] (a) Enterococcus hirae ZYpet-011, deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 32007, deposit date September 20, 2024;

[0011] (b) Turmeric and / or turmeric extract.

[0012] Furthermore, the effective dose of Enterococcus haematobacterium ZYpet-011 in the composition is: viable count of [missing information]. CFU / kg body weight / day

[0013] Furthermore, in the composition, the effective dose of turmeric or turmeric extract is 5-200 mg / kg body weight / day.

[0014] Furthermore, the 16S rRNA gene sequence of the Enterococcus haematobacterium ZYpet-011 is shown in SEQ ID NO.: 1.

[0015] Furthermore, the turmeric is dried turmeric root powder; the turmeric extract is an extract obtained by extracting turmeric as raw material with ethanol, water or a mixed solvent.

[0016] The second aspect of this invention provides the application of the Enterococcus haematobacterium and turmeric composition described in the first aspect in the preparation of pet weight management products.

[0017] Furthermore, the weight management products include pet food additives, pet nutritional supplements, pet food, and pet medications.

[0018] Furthermore, the weight management product includes one or more of the following:

[0019] (1) The product can reduce the weight of pets, liver organ index and epididymal fat organ index;

[0020] (2) The product can reduce the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in pet serum, and increase the level of high-density lipoprotein cholesterol;

[0021] (3) The product can regulate the levels of leptin and adiponectin in pet serum and improve lipid metabolism disorder.

[0022] (4) The product can reduce the levels of pro-inflammatory factors IL-6 and TNF-α in pet serum, increase the levels of anti-inflammatory factors IL-10 and IL-4, and alleviate obesity-related chronic inflammation;

[0023] (5) The product can reduce the levels of liver function-related enzymes, including γ-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, in the pet's serum, reduce liver lipid deposition and hepatocyte damage, and protect liver function.

[0024] Furthermore, the pets include pet cats and pet dogs.

[0025] A third aspect of the present invention provides a non-therapeutic method for pet weight management, the method comprising: feeding the pet the Enterococcus helicobacter pylori and turmeric composition described in the first aspect.

[0026] Information on strain preservation:

[0027] Enterococcus hirae ZYpet-011, deposited by: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Date of deposit: September 20, 2024; Accession number: CGMCC No. 32007; Classification: Enterococcus hirae.

[0028] The advantages of this invention compared to the prior art are as follows:

[0029] 1. The Enterococcus hydatidae and turmeric composition described in this application uses Enterococcus hydatidae ZYpet-011 derived from the intestines of healthy cats. It has good acid resistance, bile salt resistance and in vitro safety, and is more adapted to the cat intestinal environment. It is more conducive to long-term survival and stable function in the cat intestine than common human or dairy product-derived strains. Furthermore, when Enterococcus hydatidae ZYpet-011 is combined with turmeric / turmeric extract, it can achieve synergistic effects through bile salt hydrolysis, cholesterol reduction, anti-inflammatory and antioxidant pathways. The effect on reducing blood lipids, improving lipid metabolism disorders and reducing liver lipid deposition is significantly better than using the strain alone or using turmeric alone.

[0030] 2. The combination of Enterococcus helixeri and turmeric described in this application can effectively reduce pet weight, body fat accumulation and liver organ index, improve blood lipid indicators such as total cholesterol, triglycerides and low-density lipoprotein cholesterol, and reduce liver enzyme levels such as GGT, AST, ALT and ALP, thereby alleviating the liver function burden caused by obesity from the root cause and achieving simultaneous weight management and liver protection.

[0031] 3. The Enterococcus helicobacter and turmeric composition described in this application can downregulate pro-inflammatory factors such as IL-6 and TNF-α, upregulate anti-inflammatory factors such as IL-10 and IL-4, and regulate leptin and adiponectin levels, thereby alleviating chronic low-grade inflammation associated with obesity and improving overall metabolic status.

[0032] 4. This application is the first to use feline Enterococcus hydadrosis combined with turmeric for the management of weight and lipid metabolism in obese cats, breaking through the limitations of traditional methods that rely on food restriction and single-component intervention, and providing a new technical approach with clear targeting and stable effects for obesity-related metabolic abnormalities in pets. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0034] Figure 1 A schematic diagram of the phylogenetic tree of feline Enterococcus haematobacterium ZYpet-011;

[0035] Figure 2 Enterococcus helixeri ZYpet-011 in containing A schematic diagram showing the activity of bile salt hydrolase on a plate.

[0036] Figure 3 The results of the hemolysis test for Enterococcus haematobacterium ZYpet-011 are shown; where A: control strain Staphylococcus aureus ATCC 29213T, and B: ZYpet-011.

[0037] Figure 4 A schematic diagram of the appearance of a powder containing a combination of feline Enterococcus haematobacterium ZYpet-011 and turmeric.

[0038] Figure 5 The table shows the changes in body weight among the high-fat diet mice. (A) represents the body weight after modeling, (B) represents the trend of body weight change during the 12-week intervention period, and (C) represents the final body weight of each group of mice at week 12. Different lowercase letters indicate significant differences between groups (p < 0.05).

[0039] Figure 6 The results of organ indices for each group of mice are shown. (A) represents the liver organ index, and (B) represents the epididymal fat organ index. Different lowercase letters indicate significant differences between groups (p < 0.05).

[0040] Figure 7 The lipid profiles and lipid metabolism hormone levels of mice in each group were compared. In this study, (A) represents total cholesterol (TC), (B) represents triglycerides (TG), (C) represents high-density lipoprotein cholesterol (HDL-C), (D) represents low-density lipoprotein cholesterol (LDL-C), (E) represents leptin, and (F) represents adiponectin. Different lowercase letters indicate significant differences between groups (p < 0.05).

[0041] Figure 8 The comparison of serum inflammatory factor levels in mice of different groups is shown in the figure. (A) represents IL-6, (B) represents TNF-α, (C) represents IL-10, and (D) represents IL-4. Different lowercase letters indicate significant differences between groups (p < 0.05).

[0042] Figure 9 Schematic diagram showing the morphological observation results of HE staining of liver tissue from each group of mice;

[0043] Figure 10 This is a schematic diagram showing the results of Oil Red O staining of liver tissue from mice in each group;

[0044] Figure 11 A graph showing the changes in serum total cholesterol (TC) and triglyceride (TG) levels in obese cats in each experimental group before intervention (0 d) and after intervention (56 d);

[0045] Figure 12 The graph shows the changes in liver function-related enzyme indicators before and after intervention in obese cats in each experimental group. (A) is gamma-glutamyl transferase (GGT), (B) is aspartate aminotransferase (AST), (C) is alkaline phosphatase (ALP), and (D) is alanine aminotransferase (ALT). Detailed Implementation

[0046] 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.

[0047] 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.

[0048] 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.

[0049] MRS medium: 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, 0.25 g manganese sulfate, dissolved in deionized water, brought to a final volume of 1 L, adjusted pH to 6.0, and autoclaved at 121°C for 15 min.

[0050] Example 1: Isolation and identification of feline Enterococcus haematobacterium ZYpet-011

[0051] 1. Isolation and purification of strains

[0052] (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 evenly spread 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.

[0053] (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 ℃ 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.

[0054] (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.

[0055] (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.

[0056] (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.

[0057]

[0058] As shown in Table 1, strain ZYpet-011 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.

[0059] 2. Strain identification

[0060] 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 1Phylogenetic analysis identified strain ZYpet-011 as Enterococcus hirae and named it Enterococcus hirae ZYpet-011.

[0061] ZYpet-011 strain 16S rRNA gene sequence:

[0062]

[0063] The specific primer sequences are as follows:

[0064] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.: 2);

[0065] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.: 3).

[0066] Example 2: In vitro tolerance evaluation of Enterococcus haematobacterium ZYpet-011

[0067] (1) Physiological and biochemical characteristics of feline Enterococcus haematobacterium ZYpet-011

[0068] Enterococcus helixeri ZYpet-011 was inoculated into MRS liquid medium and cultured at 37°C. The bacterial suspension was then adjusted to OD600 = 0.8 and 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).

[0069] 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 helioris ZYpet-011 under various experimental conditions.

[0070]

[0071] As shown in Table 2, Enterococcus helixolus ZYpet-011 exhibits very weak growth at 5°C, but shows significant growth at 10°C, 45°C, and 50°C, demonstrating 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 does not grow 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 some tolerance to strongly acidic conditions.

[0072] (2) Simulated artificial gastrointestinal fluid experiment of Enterococcus hesperidinus ZYpet-011

[0073] To evaluate the survival ability of Enterococcus haematobacterium ZYpet-011 isolated in Example 1 in the gastrointestinal environment, a tolerance test was conducted using in vitro simulated gastric and intestinal fluids, as detailed below:

[0074] (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.

[0075] (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.

[0076] (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 haematobacterium* ZYpet-011 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 to calculate the survival rate of the strain in simulated gastric and intestinal fluids. All experiments were performed in triplicate to ensure data reliability.

[0077]

[0078] The results are shown in Table 3. Enterococcus hesperidinus ZYpet-011 showed good survival ability in simulated gastrointestinal fluid: its concentration in gastric fluid decreased from 9.00 CFU / mL at 0 h to 7.33 CFU / mL at 3 h, and in intestinal fluid it decreased from 6.33 CFU / mL at 0 h to 5.61 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.

[0079] Example 3: Evaluation of the in vitro lipid-lowering function of Enterococcus helixeri ZYpet-011

[0080] (1) Bile salt hydrolase experiment of Enterococcus haematobacterium ZYpet-011

[0081] 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 by hydrolysis, 500 strains of lactic acid bacteria were 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 the screening criterion. This precipitate ring was approximately 2 mm wide, with blurred edges, irregular shape, and a misty appearance surrounding the colony, showing a clear contrast to the transparent background of the culture medium.

[0082] 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.

[0083] The results are as follows Figure 2 As shown, Enterococcus helixietus ZYpet-011 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.

[0084]

[0085] Table 4 shows that the bile salt hydrolysis rate of Enterococcus haematobacterium ZYpet-011 was 75.45%, indicating that this strain has high bile salt hydrolysis activity.

[0086] (2) Evaluation of cholesterol degradation activity of Enterococcus hesperidin ZYpet-011

[0087] 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 culture at 37°C for 48 h, the culture 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 to convert the cholesterol removal rate of Enterococcus helixietus ZYpet-011.

[0088]

[0089] The results are shown in Table 5. The cholesterol removal rate of Enterococcus heliolitus ZYpet-011 was 63.29%, indicating that this strain has a strong cholesterol removal ability in vitro.

[0090] Example 4: In vitro safety evaluation of Enterococcus heyeri ZYpet-011

[0091] To investigate the safety of the bacterium isolated in Example 1—Enterococcus haematobacterium ZYpet-011—the following experiments were conducted in this example:

[0092] (1) Antibiotic susceptibility testing

[0093] The susceptibility of 10 common antibiotics to strain ZYpet-011 was determined according to the Clinical and Laboratory Standards Institute (CLSI) method. These included gentamicin (GEN), ciprofloxacin (CIP), ceftriaxone (CTR), erythromycin (E), ampicillin (AMP), tetracycline (TET), sulfamethoxazole-trimethoprim (SXT), chloramphenicol (C), lincomycin (MY), and penicillin (PEN).

[0094] A 5% inoculum of Enterococcus haematobacterium ZYpet-011 was added to MRS solid medium cooled to about 45°C. Antibiotic susceptibility testing strips were placed on the surface. The strain cultured on antibiotic-free MRS agar plates was used as a control. After incubation at 37°C for 24 h, the diameter of the inhibition zone was measured using calipers. The sensitivity of strain ZYpet-011 to 10 antibiotics was determined based on the diameter.

[0095]

[0096] As shown in Table 6, strain ZYpet-011 is resistant to ciprofloxacin and compound sulfamethoxazole, sensitive to gentamicin, erythromycin, ampicillin, chloramphenicol and penicillin, and moderately resistant to ceftriaxone, tetracycline and lincomycin.

[0097] (2) Hemolysis test

[0098] According to the instructions for Columbia blood agar plate analysis, the hemolytic activity of strain ZYpet-011 was analyzed. After two subcultures, single colonies of strain ZYpet-011 were streaked onto blood agar plates. The plates were then anaerobically cultured at 37°C for 24 hours to observe hemolytic activity, using Staphylococcus aureus as a sample. As a positive control.

[0099] like Figure 3 As shown, Staphylococcus aureus It exhibits significant hemolytic activity; the blood agar plates containing the colonies are transparent, while strain ZYpet-011 does not show hemolytic activity.

[0100] Example 5: Preparation of Enterococcus helixiez ZYpet-011 bacterial powder

[0101] This embodiment provides a method for preparing Enterococcus haematobacterium ZYpet-011 inoculum, the specific steps of which are as follows:

[0102] (1) Enterococcus heyeri ZYpet-011 was activated on MRS plate medium, and then inoculated into MRS liquid medium at an inoculation rate of 1% and cultured at 37℃ for 24 h to obtain bacterial culture.

[0103] (2) Centrifuge the bacterial culture, collect the bacterial cells, wash with sterile physiological saline, and resuspend in 15% (w / w) reconstituted skim milk to obtain a suspension. Adjust the effective viable bacterial concentration of the suspension to... CFU / mL was used to obtain a bacterial suspension. After freeze-drying the suspension, Enterococcus helixietus ZYpet-011 bacterial powder was obtained. The total number of bacteria in the powder was [missing information]. CFU / g.

[0104] Example 6: Preparation of a powder containing a combination of Enterococcus helixiezi ZYpet-011 and turmeric

[0105] This embodiment provides a composition of Enterococcus haematobacterium and turmeric for pet weight management. The preparation method of the composition includes:

[0106] (1) Raw material selection: Enterococcus haematobacterium ZYpet-011 bacterial powder obtained in Example 5 and commercially available turmeric powder that meets feed grade standards (the raw material is dried turmeric root and stem, which is obtained by crushing) were selected as raw materials for the composition.

[0107] (2) According to the proportion and mixing:

[0108] Weigh the following components according to the following mass ratio: 50 parts of Enterococcus hydatids ZYpet-011 bacterial powder; 50 parts of turmeric powder. Add the two powders to a mixer under clean conditions and mix at room temperature for 5 minutes to ensure that the bacterial powder and turmeric powder are fully and evenly mixed, thus obtaining a feline Enterococcus hydatids ZYpet-011 and turmeric combination powder.

[0109] (3) Product description: The viable count of Enterococcus haematobacterium ZYpet-011 bacterial powder Based on CFU / g, the total number of bacteria in the obtained powdered composition is approximately CFU / g, with a turmeric powder mass fraction of approximately 50 wt%.

[0110] The prepared composition powder is as follows Figure 4 As shown, the powdered composition can be further added as needed to functional pet foods or nutritional supplements for obese cats, such as cat food, nutritional pastes, etc.

[0111] Example 7: Application of the composition in a high-fat diet mouse obesity model

[0112] (1) Laboratory animals and grouping

[0113] Forty-eight SPF-grade male C57BL / 6J mice, 6 weeks old and weighing (20.0±2.0) g, were randomly divided into six groups of eight mice each. Mice were acclimatized for 7 days prior to the experiment under the following conditions: temperature (22±2)℃, relative humidity (50±10)%, and a 12-hour light / dark cycle. They had free access to standard feed and water. All experimental procedures complied with animal ethics guidelines and were approved by the animal ethics committee of the institution (Aw42405202-1-10). The modeling phase lasted 8 weeks. During this period, the high-fat model group and all intervention groups were continuously fed a high-fat diet to induce an obesity phenotype; the control group was fed a basal diet throughout. After the modeling period, a 12-week intervention period was initiated. All groups maintained their original diet regimen and received corresponding gavage treatment once daily, with a dosage of 0.2 mL per mouse. During the intervention period, the body weight, food intake, and general mental status of the mice in each group were recorded regularly. The experimental groups are as follows:

[0114] ① Control group: fed with basal feed;

[0115] ② Model group: High-fat diet + 0.2 mL physiological saline;

[0116] ③ Positive control group (Orlistat): high-fat diet + 10 mg / kg orlistat (dissolved in 0.2 mL physiological saline);

[0117] ④ZYpet-011 group (Probiotics): High-fat diet + 0.2 mL Enterococcus haematobacterium suspension ( (CFU / mL)

[0118] ⑤ Curcumin group: high-fat diet + 0.2 mL curcumin suspension (20 mg / mL);

[0119] ⑥ Combination group (Pro+Cur): High-fat diet + 0.1 mL Enterococcus hesperidin suspension ( CFU / mL) + 0.1 mL turmeric suspension (20 mg / mL);

[0120] The turmeric suspension is an aqueous solution of turmeric powder, prepared by thoroughly mixing turmeric powder and water before use.

[0121] (2) Detection indicators and methods

[0122] ① Body weight and organ index determination: During the experiment, the body weight of mice in each group was measured at fixed times each week, and body weight change curves were plotted. During the modeling period, the model was considered successfully established when the body weight of the high-fat model group exceeded the average body weight of the control group by more than 20% at the same time point. At the end of the experiment, mice were sacrificed after fasting for 12 hours, and the liver and epididymal adipose tissue were quickly removed, rinsed with PBS, dried, and weighed. The liver index and epididymal fat index were calculated.

[0123] Liver index = (liver mass / final body weight) × 100%;

[0124] Epididymal fat index = Epididymal fat mass / Final body weight × 100%.

[0125] ② Blood lipids and lipid metabolism hormones: After fasting for 12 hours at the end of the intervention, blood was collected from the orbital cavity or heart, and the serum was separated by centrifugation after standing. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured using a fully automated biochemical analyzer and matching kits; serum leptin and adiponectin concentrations were detected using commercially available ELISA kits.

[0126] ③Inflammatory factors: Serum levels of IL-6, TNF-α, IL-10 and IL-4 were measured using an ELISA kit, following the instructions. The results were used to evaluate the systemic inflammatory status and the anti-inflammatory / pro-inflammatory balance.

[0127] ④ Liver tissue pathological observation: After euthanizing the mice, left lobe liver tissue was harvested. A portion was fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned for HE staining to observe hepatic cord structure, fatty degeneration, and inflammatory cell infiltration. Another portion of liver tissue was used to prepare frozen sections, stained with Oil Red O, to evaluate the degree of lipid droplet accumulation in the liver, and semi-quantitative statistical analysis was performed using image analysis software.

[0128] like Figure 5 , Figure 6As shown, after the modeling period, the average weight of mice in the high-fat model group was 35.7 g, significantly higher than that of the control group (25.1 g) (p < 0.001), an increase of approximately 42.2%, indicating successful model establishment. After 12 weeks of intervention, the model group reached a weight of 37.0 g, significantly higher than other groups; the orlistat group (31.3 g), probiotic group (32.3 g), turmeric group (33.7 g), and combination group (33.3 g) were all significantly lower than the model group (p < 0.05), but still higher than the control group (25.7 g). Regarding organ indices, the liver index of the model group was 5.81, significantly higher than that of the control group (3.48) (p < 0.05); the orlistat group (3.82) was close to the normal level, while the probiotic group (4.36), turmeric group (5.02), and combination group (4.75) were all significantly lower than the model group (p < 0.05). In the epididymal fat index, the model group was 0.05, the control group was 0.01, the orlistat group was 0.02, the probiotic group and the combination group were both 0.04, and the turmeric group was 0.05.

[0129] like Figure 7 As shown, serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels were significantly elevated in the model group, while high-density lipoprotein cholesterol (HDL-C) levels were significantly decreased. Simultaneously, leptin levels increased and adiponectin levels decreased significantly, indicating lipid metabolism disorder. After intervention, blood lipids improved significantly in all groups, with the orlistat and probiotic groups showing outstanding lipid-lowering effects. The combined treatment group showed the most significant effect in reducing TC, TG, and LDL-C, while HDL-C levels significantly increased, with some indicators approaching those of the positive control group. Leptin levels decreased from approximately 62 pg / mL to around 40 pg / mL, and adiponectin levels rebounded from 8.76 pg / mL to 14 pg / mL, demonstrating that the combined lipid-regulating and hormone-regulating effects were superior to those of the single intervention.

[0130] like Figure 8 As shown, in the high-fat model group, the levels of pro-inflammatory factors IL-6 and TNF-α increased to 167.75 pg / mL and 62.40 pg / mL, respectively, nearly twice that of the control group (86.04 pg / mL and 29.75 pg / mL) (p < 0.05); the level of anti-inflammatory factor IL-10 decreased to 8.76 pg / mL. After intervention, the levels of TNF-α in the orlistat group and the combination group decreased to 40.37 pg / mL and 38.53 pg / mL, respectively, representing the two groups with the largest decreases; the turmeric group and the probiotic group also showed some inhibitory effects. In the combination group, IL-10 rebounded to around 14 pg / mL, and IL-4 also showed a significant increase, indicating a significant relief of the inflammatory state.

[0131] like Figure 9 , Figure 10As shown, HE staining revealed that the control group had normal hepatocyte structure; the model group exhibited disordered hepatic cord arrangement, abundant lipid droplet accumulation, and inflammatory infiltration, showing significant fatty degeneration. The orlistat group showed reduced hepatocyte degeneration and fewer lipid droplets; the probiotic and turmeric groups also showed reduced lipid droplets and inflammation. The combined group showed near-complete restoration of hepatic cord structure, a significant reduction in the area and number of lipid droplets, and very mild inflammatory infiltration. Oil Red O staining results were consistent with these findings, with the combined group showing a significantly smaller red-stained area than the model group, indicating a significant reduction in intrahepatic lipid deposition.

[0132] Example 8: Application of the combination of Enterococcus hesperidin and turmeric in obese cats

[0133] To investigate the role of the combination of Enterococcus hesperidin and turmeric in pet weight management, this example uses a pet cat as an example and conducts the following animal experiment:

[0134] (1) Experimental Animals and Grouping: Twenty-four adult pet cats aged 15–18 months with a Body Condition Score (BCS, 9-point scale) greater than 7 and a tendency towards high blood lipids 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 might affect the gut microbiota within at least one month. The experimental protocol was approved by the animal ethics committee. The experimental environment adopted a 12-hour light / 12-hour dark cycle, with room temperature controlled at 18–26 ℃ and relative humidity at 40%–70%. The cat houses were cleaned twice daily and disinfected once daily to maintain environmental hygiene. Each group of cats was fed twice daily with a fixed amount of about 35 g per cat, and had free access to water. A one-week adaptation period was set before the intervention, during which each group was fed only a basic diet without the addition of functional ingredients. The formal intervention then began for 56 days, with probiotics, turmeric, or a combination of these added to the same basic diet.

[0135] After being evenly divided by weight and BCS, the animals were randomly divided into 4 groups of 6 each, as follows:

[0136] ① Control group: Basic cat food;

[0137] ② Probiotics Group: Basic cat food + feline-derived Enterococcus haematobacterium ZYpet-011 freeze-dried probiotic powder, supplemented daily with 10 mg / kg of Enterococcus haematobacterium probiotic (the probiotic contains approximately 10 mg / kg of live bacteria). CFU / g);

[0138] ③ Turmeric group (Curcumin): Basic cat food + 10 mg / kg of turmeric powder per day based on body weight;

[0139] ④ Combination Group (Pro+Cur): Basic cat food + 5 mg / kg of Enterococcus haematobacterium inoculant (the inoculant contains approximately 100 live bacteria). CFU / g) and 5 mg / kg turmeric powder.

[0140] (2) Detection indicators: Before the intervention (day 0) and at the end of the intervention (day 56), cats in each group were fasted for 12 hours at night but had free access to water. Blood was collected from the forelimb veins the next day, with approximately 2 mL of whole blood collected from each cat. Part of the blood sample was placed in EDTA anticoagulant tubes for routine hematological testing; the other part was placed in serum separation tubes without anticoagulant, allowed to stand at 28°C to coagulate, and then centrifuged at 3000×g for 15 min at 4°C. The serum was separated and stored at -80°C for later testing. The following indicators closely related to obesity and lipid metabolism abnormalities were measured using an automated animal blood biochemistry analyzer: lipid indicators: total cholesterol (TC), triglycerides (TG); liver function and fatty liver-related enzyme indicators: gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Statistical software was used to analyze the indicators, compare the changes in each group on day 0 and day 56, and evaluate the effects of different intervention methods on improving obesity-related dyslipidemia and liver function damage.

[0141] The results are as follows Figure 11 As shown, the serum total cholesterol (TC) and triglyceride (TG) levels in the control group showed no significant changes (ns) between day 0 and day 56, indicating that maintaining the original feeding management alone was insufficient to significantly improve blood lipids. In the probiotic group, turmeric group, and combined treatment group, TC levels significantly decreased from baseline after 56 days of intervention (p < 0.001), indicating that all three intervention methods had a clear cholesterol-lowering effect. Regarding TG, there was no significant difference (ns) between day 0 and day 56 in the control group and turmeric group, while the TG levels in the probiotic group and the combined treatment group were significantly lower on day 56 than on day 0 (p < 0.01), suggesting that feline probiotics had a more prominent regulatory effect on TG, and the combined treatment also showed a significant reduction in TG. Overall, the combined treatment group showed the most comprehensive performance in simultaneously reducing TC and TG, possessing both the TG-lowering advantage of probiotics and the TC-lowering effect of turmeric, resulting in a better overall lipid-lowering effect than either the probiotic group or the turmeric group alone.

[0142] Figure 12The study showed the effects of the combination on liver function enzyme indicators, including gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). In the control group, none of these indicators showed significant changes (ns) between 0 and 56 days, suggesting that basal feeding alone had a limited effect on improving obesity-related liver burden. In contrast, the levels of GGT, AST, and ALT in the probiotic group, turmeric group, and combination group were significantly lower on day 56 than on day 0 (p < 0.01), indicating that all three interventions could alleviate hepatocellular damage to varying degrees. The combination group showed a more consistent and greater decrease in all enzyme indicators, demonstrating a better overall improvement than the individual groups. Regarding ALP levels, the probiotic group and the combination group showed a significant decrease on day 56 (p < 0.01), and the turmeric group also showed a certain decrease (p < 0.05), indicating that the probiotics and the combination had a more prominent effect on alleviating cholestasis and hepatobiliary burden. Based on the combined results of blood lipids and liver enzymes, the probiotic + turmeric combination showed a better overall effect of "lipid reduction + liver protection" in obese cats than either probiotics or turmeric alone.

[0143] 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. The use of the composition in the preparation of a weight management medicine for pet cats or dogs, characterized in that, The composition consists of Enterococcus hirae ZYpet-011 and turmeric. composition: (a) Enterococcus heyeri ZYpet-011, deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 32007, deposit date September 20, 2024; (b) The turmeric mentioned is dried turmeric root powder; In the composition, the effective dose of Enterococcus haematobacterium ZYpet-011 is: viable count of CFU / kg body weight / day; the effective dose of turmeric is 5 mg / kg body weight / day.

2. The application according to claim 1, characterized in that, The 16S rRNA gene sequence of Enterococcus haematobacterium ZYpet-011 is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The weight management medication is one or more of the following: (1) The drug can reduce the weight of pets, liver organ index and epididymal fat organ index; (2) The drug can reduce the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in pet serum, and increase the level of high-density lipoprotein cholesterol; (3) The drug can regulate the levels of leptin and adiponectin in pet serum and improve lipid metabolism disorder. (4) The drug can reduce the levels of pro-inflammatory factors IL-6 and TNF-α in pet serum, increase the levels of anti-inflammatory factors IL-10 and IL-4, and alleviate obesity-related chronic inflammation; (5) The drug can reduce the levels of liver function-related enzymes, including γ-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, in the pet's serum, reduce liver lipid deposition and hepatocyte damage, and protect liver function.

Images