Bacillus subtilis metabalance bs01 for relieving obesity of animals and application thereof

Abstract

The application discloses a bacillus subtilis Metabalance BS01 for relieving animal obesity and an application thereof. The Metabalance BS01 is preserved in the China Center for Type Culture Collection, and the preservation date is June 10, 2026, and the preservation number is CCTCC NO: M 20261252. The number of live bacteria in a probiotic preparation is (1-6) × 10 9 CFU / g, and the preparation is a freeze-dried powder or a microcapsule powder. The bacillus subtilis Metabalance BS01 provided by the application can significantly reduce the body weight of adult obese cats and the serum total cholesterol content of the adult obese cats, can significantly reduce the body weight and adipose tissue weight of HFD-induced obese mice, and can significantly reduce the serum triglyceride and total cholesterol content of the obese mice. The above results indicate that the bacillus subtilis Metabalance BS01 can be used as an animal feed additive, can relieve the obesity of adult cats, mice and other animals, can improve lipid metabolism disorder, and has a great application prospect in the fields of pet, livestock and poultry health and efficient breeding, and regulation of lipid metabolism disorder.

Description

Technical Field

[0001] This invention relates to the fields of feed and veterinary drug technology, specifically to a strain of Bacillus subtilis Metabalance BS01 that alleviates obesity in adult cats and induces obesity in mice with HFD, and its applications. Background Technology

[0002] When the body's energy intake and expenditure are imbalanced, excessive fat accumulation occurs, leading to metabolic diseases such as obesity. In recent years, with changes in lifestyle and dietary structure, the incidence of obesity has continued to rise, seriously endangering not only human health but also showing a high prevalence in companion animals such as cats and dogs. Obesity can cause various complications such as glucose and lipid metabolism disorders, insulin resistance, and cardiovascular diseases, severely impacting animal health and lifespan. Therefore, developing safe and effective intervention measures for animal obesity is of great significance.

[0003] Currently, diet control, exercise management, and drug therapy are the main methods for alleviating obesity. However, long-term dietary restriction and increased exercise are extremely challenging for both humans and pets, making them difficult to maintain in practice, and some weight-loss drugs may have side effects with long-term use. Recent studies have found that probiotics and their metabolites can play a positive role in alleviating obesity and improving metabolic disorders by regulating gut microbiota structure and host metabolism. Existing research has shown that some Bacillus subtilis and compound probiotic preparations can, to some extent, inhibit weight gain induced by a high-fat diet and improve lipid metabolism indicators.

[0004] Currently, the application of specific Bacillus subtilis strains in alleviating animal obesity remains relatively limited, particularly lacking research reports on their effects in alleviating animal obesity, inhibiting fat deposition, and improving obesity-related metabolic disorders. Therefore, developing Bacillus subtilis strains with anti-obesity effects has significant application value in the field of animal nutrition and health. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a strain of Bacillus subtilis Metabalance BS01 that alleviates obesity in animals and its applications.

[0006] The technical solution of the present invention is as follows: A strain of Bacillus subtilis Metabalance BS01 that alleviates obesity in animals has been deposited at the China Center for Type Culture Collection (CCTCC) on June 10, 2026, with accession number CCTCC NO: M20261252.

[0007] The probiotic preparation contains (1-6) × 10⁻⁶ live bacteria. 9 The dosage form is CFU / g, and the preparation is a lyophilized powder or a microcapsule powder.

[0008] The preparation method of the probiotic preparation includes the following steps: Step S1: Inoculating the Bacillus subtilis Metabalance BS01 into LB medium and activating it to the late logarithmic growth stage; Step S2: Collecting the bacterial cells by centrifugation, washing, and adding a cryoprotectant for suspension mixing; Step S3: Freeze-drying to obtain probiotic powder; the viable count of the Bacillus subtilis Metabalance BS01 strain is 4.5 × 10⁻⁶. 9 CFU / g.

[0009] In the preparation method described above, the inoculum amount of Bacillus subtilis Metabalance BS01 strain is 0.5%-5% (V / V).

[0010] The cryoprotectant formulation, by weight percentage, consists of: 10% trehalose, 5% skim milk powder, and 2% glycerin, with the remainder being sterile water.

[0011] The application of Bacillus subtilis Metabalance BS01 in the preparation of probiotic preparations, functional products, animal feed additives, or veterinary drugs for alleviating obesity in adult cats, HFD-induced obesity in mice, and regulating lipid metabolism.

[0012] The method of application includes: diluting the Bacillus subtilis preparation to a viable count of (1-6)×10⁻⁶. 9 CFU / mL, administered orally or mixed with feed to adult obese cats or HFD-induced obese mice, 1-3 times daily for 3-8 weeks.

[0013] The Bacillus subtilis was diluted with LB medium to a viable cell concentration of 1×10⁻⁶. 9 CFU / mL bacterial suspension, per Only For adult obese cats, the gavage dose is 2 mL, once daily, for 4 consecutive weeks.

[0014] The Bacillus subtilis was diluted with LB medium to a viable cell concentration of 1×10⁻⁶. 9 The bacterial suspension at a concentration of CFU / mL was administered to each obese mouse via gavage at a dose of 0.2 mL, once daily for 3 consecutive weeks.

[0015] The relief of adult feline obesity refers to: reducing the cat's body weight and reducing serum total cholesterol (TChol) and low-density lipoprotein cholesterol (LDL-C) levels; the relief of HFD-induced mouse obesity refers to: reducing the body weight, inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) weight of obese mice, and reducing serum triglyceride (TG) and total cholesterol (TChol) levels.

[0016] The beneficial effects of this invention are: In an experiment on obese adult cats, after continuous gavage administration of this strain, the cats' body weight decreased significantly from day 21, and obesity-related lipid metabolism indicators improved, indicating that this strain has an effect in alleviating obesity.

[0017] In a high-fat diet-induced obese mouse model, continuous gavage administration of Bacillus subtilis significantly reduced body weight and adipose tissue (including inguinal and epididymal white fat) weight, and dramatically reduced serum triglycerides and cholesterol. This further demonstrates that the bacterium has a good effect in alleviating obesity, reducing fat deposition, and improving lipid metabolism disorders.

[0018] In summary, the Bacillus subtilis Metabalance BS01 developed in this invention has promising application prospects in animal weight loss products, pet functional products, and feed additives. Attached Figure Description

[0019] Figure 1 This is a distribution map of the whole genome sequence of Bacillus subtilis Metabalance BS01.

[0020] Figure 2 The results are from the hemolysis test of Bacillus subtilis Metabalance BS01.

[0021] Figure 3 The effect of gavage administration of Bacillus subtilis Metabalance BS01 to experimental cats on the body weight of adult obese cats. Significant differences are indicated by * (P < 0.05).

[0022] Figure 4 The figure shows the effect of gavage administration of LB medium to control cats on the body weight of obese adult cats.

[0023] Figure 5 The effect of gavage administration of Bacillus subtilis Metabalance BS01 on serum total cholesterol levels in adult obese cats is shown in the figure. Significant differences are indicated by * (P < 0.05).

[0024] Figure 6 The effect of gavage administration of Bacillus subtilis Metabalance BS01 on serum low-density lipoprotein cholesterol levels in adult obese cats is shown in the figure. Significant differences are indicated by * (P < 0.05).

[0025] Figure 7 Figure showing the effect of gavage administration of Bacillus subtilis Metabalance BS01 on the body weight of obese mice induced by a high-fat diet. Extremely significant differences are indicated by ** (P < 0.01).

[0026] Figure 8The figure shows the effect of gavage administration of Bacillus subtilis Metabalance BS01 on body weight changes in obese mice induced by a high-fat diet. Significant differences (P < 0.05) are indicated by *, and highly significant differences (P < 0.01) are indicated by **.

[0027] Figure 9 Figure showing the effect of gavage administration of Bacillus subtilis Metabalance BS01 on the weight of white adipose tissue in the groin of obese mice induced by a high-fat diet. Extremely significant differences are indicated by ** (P < 0.01).

[0028] Figure 10 Figure showing the effect of gavage administration of Bacillus subtilis Metabalance BS01 on the weight of white adipose tissue in the epididymis of obese mice induced by a high-fat diet. Extremely significant differences are indicated by ** (P < 0.01).

[0029] Figure 11 The graph shows the effect of gavage administration of Bacillus subtilis Metabalance BS01 on serum triglyceride levels in obese mice induced by a high-fat diet. Extremely significant differences are indicated by ** (P < 0.01).

[0030] Figure 12 The graph shows the effect of gavage administration of Bacillus subtilis Metabalance BS01 on serum cholesterol levels in obese mice induced by a high-fat diet. Extremely significant differences are indicated by ** (P < 0.01). Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0032] Safety assessments of Bacillus subtilis Metabalance BS01 were conducted prior to the obesity intervention trial. Hemolytic activity tests were negative (see...). Figure 2 ). Example 1

[0033] Preparation of probiotic formulations to alleviate obesity in adult cats and mice 1) Inoculate Bacillus subtilis Metabalance BS01 strain into LB medium and culture it in a conventional bacterial incubator to obtain fresh bacterial culture; 2) The inoculation amount of the Bacillus subtilis Metabalance BS01 strain was 2% (V / V, 2% by volume) into LB medium to obtain fresh bacterial culture; 3) Transfer the liquid culture medium to a sterile centrifuge tube and centrifuge at 8000 r / min for 10 minutes at 25°C. Remove the supernatant, collect the precipitate, wash once with PBS, and add the prepared cryoprotectant (cryoprotectant formula: 10% trehalose, 5% skim milk powder and 2% glycerol, the remainder being sterile water) at a ratio of 1:1. Vortex to mix well to obtain a bacterial suspension, ensuring that the cryoprotectant and bacterial cells are fully integrated. 4) Pre-freeze the well-mixed freeze-dried bacterial suspension in a -80℃ freezer for approximately 24 hours. Turn on the freeze dryer 20 minutes beforehand to lower the cold trap temperature to -55℃ to prevent damage to the bacteria from a sudden temperature rise. Immediately after removing the bacterial suspension, transfer it to the freeze dryer for freeze-drying for approximately 32 hours. After freeze-drying, quickly remove the sample. The viable count of Bacillus subtilis Metabalance BS01 strain in the freeze-dried bacterial powder is approximately 4.5 × 10⁻⁶. 9 CFU / g.

[0034] 5) Dissolve the prepared probiotic powder in the culture medium at a 1:1 ratio, mix well, and the viable bacteria content in the bacterial solution should be 10. 9 CFU / mL; Discard the needle in a 2.5mL syringe and draw 2 mL for gavage. Example 2

[0035] Strain identification like Figure 1 As shown, whole-genome sequencing was performed on this strain of Bacillus subtilis, and species annotation analysis was conducted on the sequencing results. The results showed that Bacillus subtilis-related annotated sequences accounted for 78.49% of all annotated sequences (…). Figure 1 Based on morphological characteristics (rod-shaped, spore-producing, Gram-positive) and biochemical identification results, it was named Bacillus subtilis Metabalance BS01. Example 3

[0036] hemolysis test Dip a sample of Bacillus subtilis Metabalance BS01 culture into Columbia blood agar solid medium and incubate at 37°C for 14-18 hours. Observe whether a hemolytic ring is formed.

[0037] The results are as follows Figure 2 As shown, hemolysis occurred around E. coli, while no changes were observed around Metabalance BS01 and PBS buffer, indicating that Bacillus subtilis Metabalance BS01 has no hemolytic effect. Figure 2). Example 4

[0038] Application of Bacillus subtilis Metabalance BS01 in adult obese cats Nine adult obese cats were selected and fed under the same conditions. Four control group adult obese cats were orally administered 2 mL of LB culture medium daily, while five experimental group adult obese cats were orally administered 2 mL of 1×10⁻⁶ medium daily. 9 Metabalance BS01 probiotic suspension (CFU / mL) was administered to cats for 28 consecutive days. Body weight was measured on days 0, 7, 14, 21, and 28; serum biochemical parameters were measured on days 0, 14, and 28.

[0039] Statistical analysis was performed using GraphPad Prism software. Paired t-tests were used for comparisons within each group. Data are expressed as mean ± standard error (SEM), and a p-value < 0.05 was considered statistically significant.

[0040] like Figure 3 , Figure 4 As shown, after 21 and 28 days of intervention with the probiotic Metabalance BS01 in this invention, the weight of the pet cats was significantly reduced compared to before the intervention (P < 0.05). Figure 3 ); No significant change in body weight was observed in adult obese cats in the gavage LB control group ( Figure 4 This indicates that the strain has a good effect in alleviating obesity.

[0041] The results of changes in serum lipid metabolism-related indicators in adult obese cats during the intervention period are shown in the figure. Figure 5 , Figure 6 After 14 days of intervention, the serum total cholesterol (TChol) level in the pet cats showed a decreasing trend. After 28 days of intervention, the total cholesterol level decreased from 2.76 ± 0.13 mmol / L to 2.40 ± 0.23 mmol / L, a decrease of 0.36 mmol / L (approximately 13.18%). Figure 5 Low-density lipoprotein cholesterol (LDL-C) was also significantly reduced. Figure 6 This indicates that Bacillus subtilis Metabalance BS01 can improve lipid metabolism disorders caused by obesity.

[0042] In summary, the Bacillus subtilis Metabalance BS01 of this invention can reduce the body weight of obese adult cats, while also reducing the levels of total cholesterol and low-density lipoprotein cholesterol in the serum of obese adult cats, thus improving lipid metabolism disorders in obese adult cats. In the parallel control group using LB medium, no significant change in body weight was observed in the obese cats. Example 5

[0043] Application of Bacillus subtilis Metabalance BS01 in high-fat diet-induced obese mice Obese mice induced by a high-fat diet for 10 weeks were used as an obesity model. The obese mice weighed 39-44g and were randomly divided into a high-fat control group (HFD group, n=5) and a probiotic treatment group (HFD+BS01 group, n=5). There was no significant difference in initial body weight between the two groups (P > 0.05). The probiotic treatment group was administered 0.2 mL of Bacillus subtilis suspension (1×10^9 CFU / mL) daily by gavage; the high-fat control group was administered 0.2 mL of LB medium daily by gavage for 21 consecutive days. Mouse body weight was measured on days 0, 7, 14, and 21. Mice were sacrificed on day 22, and samples were taken. Inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) were separated, weighed, and the data were recorded.

[0044] Statistical analysis was performed using GraphPad Prism software. Independent samples t-tests were used to compare the two groups. Data are expressed as mean ± standard error (SEM), and p < 0.05 was considered statistically significant.

[0045] Results of mouse weight changes are shown in Figure 7 , Figure 8 On days 14 and 21 after intervention, the body weight of mice in the BS01 probiotic treatment group was significantly lower than that in the high-fat control group (P < 0.01 or P < 0.001). Figure 7 Weight changes began on day 7, with the probiotic treatment group showing significantly greater weight changes than the high-fat control group. Figure 8 This indicates that this strain of Bacillus subtilis has a significant effect in reducing obesity and weight gain induced by a high-fat diet in mice.

[0046] The weight of adipose tissue in the two groups of mice after intervention is shown in the figure. Figure 9 , Figure 10 Compared with the high-fat control group, mice in the BS01 probiotic treatment group had significantly higher levels of inguinal white adipose tissue (iWAT). Figure 9 ) and epididymal white fat (eWAT, Figure 10 The weight of the samples was significantly reduced (P < 0.05 or P < 0.01), indicating that the strain can significantly reduce fat deposition and alleviate obesity.

[0047] like Figure 11 , Figure 12 As shown, Bacillus subtilis in this invention significantly improves lipid metabolism disorders induced by HFD in obese mice, specifically: compared with the control group, probiotic treatment significantly reduced the levels of triglycerides and total cholesterol in the serum of obese mice (P < 0.01). Figure 11, Figure 12 ).

[0048] In summary, the probiotic preparation of this invention can treat HFD-induced obesity in mice, reduce the body weight and adipose tissue weight of high-fat diet-induced obese mice, and significantly reduce serum triglyceride and total cholesterol levels in obese mice, thus improving lipid metabolism disorders in obese mice. This further demonstrates that the probiotic has an effect in alleviating animal obesity.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the concept and scope of the invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalent technical solutions.

Claims

1. A strain of Bacillus subtilis Metabalance BS01 that alleviates obesity in animals, characterized in that, It is deposited at the China Center for Type Culture Collection on June 10, 2026, with accession number CCTCC NO:M 20261252.

2. A probiotic preparation containing Bacillus subtilis Metabalance BS01 as described in claim 1, characterized in that: The probiotic preparation contains (1-6) × 10⁻⁶ live bacteria. 9 The dosage form is CFU / g, and the preparation is a lyophilized powder or a microcapsule powder.

3. A method for preparing the probiotic preparation according to claim 2, characterized in that: Including the following Steps: Step S1: Inoculate the Bacillus subtilis Metabalance BS01 into LB medium and activate it to the late logarithmic growth stage; Step S2: Collect the bacterial cells by centrifugation, wash them, and add a cryoprotectant for resuscitation and mixing; Step S3: Freeze-dry to obtain probiotic powder; The viable count of the Bacillus subtilis Metabalance BS01 strain is 4.5 × 10⁻⁶. 9 CFU / g.

4. The preparation method according to claim 3, characterized in that, The inoculation amount of the Bacillus subtilis Metabalance BS01 strain is 0.5%-5% (V / V).

5. The preparation method according to claim 3, characterized in that: The cryoprotectant formulation, by weight percentage, consists of: 10% trehalose, 5% skim milk powder, and 2% glycerin, with the remainder being sterile water.

6. The application of Bacillus subtilis according to claim 1 or the probiotic preparation according to claim 2, characterized in that, The application of Bacillus subtilis Metabalance BS01 in the preparation of probiotic preparations, functional products, animal feed additives, or veterinary drugs for alleviating obesity in adult cats, HFD-induced obesity in mice, and regulating lipid metabolism.

7. The application according to claim 6, characterized in that: The method of application includes: diluting the Bacillus subtilis preparation to a viable count of (1-6)×10⁻⁶. 9 CFU / mL, administered orally or mixed with feed to adult obese cats or HFD-induced obese mice, 1-3 times daily for 3-8 weeks.

8. The application according to claim 7, characterized in that, The Bacillus subtilis was diluted with LB medium to a viable cell concentration of 1×10⁻⁶. 9 CFU / mL bacterial suspension, per Only For adult obese cats, the gavage dose was 2 mL, once daily, for 4 consecutive weeks.

9. The application according to claim 7, characterized in that, The Bacillus subtilis was diluted with LB medium to a viable cell concentration of 1×10⁻⁶. 9 The bacterial suspension at a concentration of CFU / mL was administered to each obese mouse via gavage at a dose of 0.2 mL, once daily for 3 consecutive weeks.

10. The application according to claim 6, characterized in that: The relief of adult feline obesity refers to: reducing the cat's body weight and reducing serum total cholesterol (TChol) and low-density lipoprotein cholesterol (LDL-C) levels; the relief of HFD-induced mouse obesity refers to: reducing the body weight, inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) weight of obese mice, and reducing serum triglyceride (TG) and total cholesterol (TChol) levels.

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