A strain of Weissella paramesenteroides WPB25, its inoculants, and their applications

Abstract

This invention relates to a plant Weissella paramesenteroides Weissella paramesenteroides WPB25, its inoculants, and their applications belong to the technical field of microbiology. Weissella paramesenteroides Weissella paramesenteroides WPB25 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37194, on December 25, 2025. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This invention also provides... Weissella paramesenteroides Weissella paramesenteroides Application of WPB25 in the preparation of products for the prevention, relief, or treatment of obesity, treatment of metabolic syndrome, relief of inflammation, and enhancement of antioxidant capacity. This invention provides... Weissella paramesenteroides WPB25 can prevent fatty liver from developing into steatohepatitis, providing a safer and more effective treatment option for obese patients.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain Weissellaparamesenteroides WPB25 and its inoculants and applications. Background Technology

[0002] Obesity has become an increasingly serious global health problem and a major risk factor for chronic diseases such as type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. With rising global obesity rates, the incidence of metabolic-associated fatty liver disease (MASLD) continues to increase, and it is closely related to the high-fat, high-sugar Western diet. MASLD encompasses liver lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and can progress to liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Currently, there are no approved drug treatments for MASLD. Existing treatments for MASLD and related metabolic disorders often have limited efficacy or significant side effects. For example, some anti-obesity drugs may cause gastrointestinal problems, increase the risk of acute pancreatitis, or may lead to an increased risk of diabetes and elevated liver transaminase levels. Therefore, there is an urgent need to develop innovative treatment strategies that are biocompatible, highly effective, and tolerable.

[0003] Furthermore, pet obesity rates are rising sharply, affecting approximately 40% to 60% of dogs. Animal obesity not only affects appearance but is also closely related to pathological conditions such as insulin resistance, dyslipidemia, hepatic steatosis, and immune dysfunction. Obesity significantly shortens pet lifespan, reduces their quality of life, and places a dual emotional and financial burden on owners. While traditional drug treatments can improve some clinical indicators, they are often limited by significant side effects, poor compliance, and uncertain long-term efficacy. These trends highlight the urgent need to develop effective, safe, and sustainable interventions for obesity in humans and companion animals.

[0004] Probiotics have shown potential in improving host metabolism by regulating the gut microbiota. Traditional strains such as Lactobacillus and Bifidobacterium can alleviate metabolic disorders through multiple mechanisms. For example, Lactobacillus rhamnosus GG can reduce oxidative stress and inflammatory responses in rats with non-alcoholic fatty liver disease; both live and heat-inactivated Bifidobacterium animalis J-12 have been shown to alleviate oral ulcers in golden hamsters by regulating the gut microbiota; and Lactobacillus plantarum FRT4 can prevent fatty liver hemorrhage syndrome by regulating the gut microbiota and the FOXO / TLR-4 / NF-κB signaling pathway. However, the effects of probiotics are highly strain-specific, and even closely related strains may have different metabolic effects. Currently, most research focuses on food or human-derived strains, with relatively limited attention paid to companion animal-derived probiotics, which limits their targeted application in pet nutrition and disease intervention. Notably, canine Lactobacillus may possess host-adaptive advantages in the specific canine gut environment, providing a more effective pathway for regulating pet metabolism and obesity.

[0005] Enterobacter vesiculosus ( Weissella paramesenteroides This species of lactic acid bacteria belongs to the Lactobacillus family and has significant potential as a probiotic in regulating lipid metabolism. No applications of *Enterobacterium vesiculosus* in regulating lipid metabolism, enhancing antioxidant capacity, and suppressing inflammation have been found to date. Summary of the Invention

[0006] To address the lack of application of *Westernella enterica* in regulating lipid metabolism, enhancing antioxidant capacity, and inhibiting inflammatory responses in existing technologies, this invention provides a strain... Weissellaparamesenteroides WPB25 and its inoculants and applications are used to address the aforementioned problems.

[0007] In a first aspect, the present invention provides a plant Weissellaparamesenteroides WPB25, the Weissella paramesenteroides WPB25 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37194, on December 25, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, the aforementioned Weissellaparamesenteroides The 16S rDNA sequence of WPB25 is shown in SEQ ID NO.1.

[0009] Secondly, the present invention provides a product containing Weissellaparamesenteroides The formulation of WPB25.

[0010] Furthermore, the bacterial agent is a liquid or solid formulation.

[0011] Furthermore, in the liquid formulation,Weissellaparamesenteroides The content of WPB25 is 1.0 × 10⁻⁶. 7 ~1.0×10 10 CFU / mL; in the solid dosage form Weissellaparamesenteroides The content of WPB25 is 1.0 × 10⁻⁶. 7 ~1.0×10 10 CFU / g.

[0012] Furthermore, the preparation method of the liquid formulation is as follows: Weissella paramesenteroides WPB25 was activated for two generations, then inoculated into MRS liquid medium at an inoculum rate of 2% (v / v) and incubated statically at 37°C for 24 hours. After incubation, the cells were collected by centrifugation at 4°C and 5000 rpm for 10 minutes, washed twice with 0.9% physiological saline, and adjusted using the plate viable count method. Weissellaparamesenteroides WPB25 has a viable bacterial count of 1×10⁻⁶. 10 CFU / mL, to obtain a concentration of Weissellaparamesenteroides WPB25 liquid bacterial agent.

[0013] Furthermore, the composition of the MRS liquid culture medium is as follows: 10.0g peptone, 1.0mL Tween 80, 4.0g yeast extract, 2.0g triammonium citrate, 0.05g manganese sulfate, 5.0g beef meal, 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 0.2g magnesium sulfate, and 5.0g sodium acetate, dissolved in 1000mL distilled water, pH=6.2±0.2; autoclaved at 121℃ for 15min.

[0014] Thirdly, the present invention provides Weissellaparamesenteroides Application of WPB25 in the preparation of products for the prevention, relief or treatment of obesity.

[0015] Fourthly, the present invention provides Weissellaparamesenteroides Application of WPB25 in the preparation of products for the prevention, relief or treatment of metabolic syndrome.

[0016] Fifthly, the present invention provides Weissellaparamesenteroides Application of WPB25 in the preparation of products that alleviate inflammation and enhance antioxidant properties.

[0017] The beneficial effects of this invention are as follows: The present invention provides Weissellaparamesenteroides WPB25 can alleviate lipid metabolism, reduce steatosis, enhance antioxidant capacity, and alleviate inflammatory responses, thus preventing fatty liver from developing into steatohepatitis and providing a safer and more effective treatment option for obese patients. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the present invention. Weissellaparamesenteroides Colony morphology diagram of WPB25.

[0020] Figure 2 This is the present invention. Weissellaparamesenteroides Microscopic image of WPB25.

[0021] Figure 3 This is a graph showing the trend of weight change in each group of mice in Example 3 of the present invention.

[0022] Figure 4 This is a bar chart of organ indexes for each group of mice in Example 3 of the present invention.

[0023] Figure 5 These are staining images of mouse liver tissues from each group in Example 3 of this invention.

[0024] Figure 6 These are staining images of mouse adipose tissue in each group in Example 3 of this invention. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0026] The various culture medium components used in the embodiments of this invention are as follows: MRS liquid culture medium: 10.0g peptone, 1.0mL Tween 80, 4.0g yeast extract, 2.0g triammonium citrate, 0.05g manganese sulfate, 5.0g beef meal, 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 0.2g magnesium sulfate, and 5.0g sodium acetate, dissolved in 1000mL distilled water, pH=6.2±0.2, autoclaved at 121℃ for 15min.

[0027] MRS solid medium: Add 2% agar powder to MRS liquid medium.

[0028] MRS-CaCO3 solid medium: Add 2% CaCO3 to MRS liquid medium.

[0029] Example 1 Weissellaparamesenteroides Isolation, screening and identification of WPB25 1. Sampling: Feces from healthy broiler chickens were collected in Beijing in June 2025.

[0030] 2. Separation and screening: (1) Collect 2g of fresh fecal sample from healthy broilers, add 18mL of sterile physiological saline, mix thoroughly on a vortex shaker, add an appropriate amount of glass beads, and place the mixture in a 37℃ shaker (200 rpm / min) for 30min.

[0031] (2) 10% serially diluted with sterile physiological saline -1 10 -2 10 -3 and 10 -4 Take 100 μL of each dilution from each gradient, spread it evenly on MRS-CaCO3 solid medium, and incubate it in a 37°C constant temperature incubator for 48 h.

[0032] (3) Select single colonies of suspected lactic acid bacteria with milky white and raised features, and perform streak purification on MRS solid medium. Repeat twice to ensure purity, and incubate in a constant temperature incubator at 37℃ for 48h.

[0033] (4) Inoculate a single colony of each isolate into MRS liquid medium and incubate at 37°C for 24 hours.

[0034] (5) Add the bacterial culture to 50% glycerol and store it in a -80°C freezer for later use.

[0035] 3. Identification (1) Identification of colony morphology When strain WPB25 was inoculated onto MRS agar medium and incubated at 37°C for 24 hours, the colonies were observed to be nearly round with neat edges, a smooth, moist, milky-white, opaque surface, moderate elevation, and uniform texture. The colony morphology of strain WPB25 is shown in the figure below. Figure 1 As shown, the optical microscope image is as follows: Figure 2 As shown.

[0036] (2) Molecular biological identification Single colonies of strain WPB25 were streaked onto NA plates and incubated at 35°C for 12 hours. The incubated NA plates were then sent to Beijing Qingke Biotechnology Co., Ltd. for strain identification. The 16S rDNA sequence is shown in SEQ ID NO.1.

[0037] After identification, the strain is... Weissellaparamesenteroides , Weissellaparamesenteroides WPB25 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37194, on December 25, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0038] 4. Identification of physiological and biochemical characteristics (1) Acid production experiment Will Weissellaparamesenteroides After WPB25 was activated for the second generation, it was transferred to 50 ml LMR syrup at a 2% inoculum and incubated statically at 37°C for 24 hours. Subsequently, it was centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected for pH detection and lactate content analysis using a lactate kit.

[0039] The results showed that after 24 hours of incubation, Weissellaparamesenteroides The pH of the culture medium for WPB25 was 3.8, which reached 3.48 after 48 hours, indicating a relatively strong acidity. At the same time, the lactic acid concentration was measured to be 48.64 μmol / mL after 48 hours.

[0040] (2) Antibiotic susceptibility test Will Weissellaparamesenteroides After two subcultures and activation, WPB25 was inoculated into MRS liquid medium at an inoculum size of 2.0% (v / v) and incubated at 37°C for 6 hours. 100 μL of the bacterial suspension was evenly spread onto plates, and antimicrobial susceptibility test discs containing different antibiotics (tetracycline 30 μg / disc, clindamycin 2 μg / disc, gentamicin 10 μg / disc, erythromycin 15 μg / disc, chloramphenicol 30 μg / disc, ampicillin 10 μg / disc) were attached to each disc. Positive control discs were also included. The plates were incubated at 37°C for another 24 hours, and the formation of inhibition zones was observed. The results were interpreted as follows: a clear inhibition zone was marked as "S" (sensitive), and no inhibition zone was marked as "R" (resistant). Detailed test results are shown in Table 1.

[0041] Table 1- Weissellaparamesenteroides antibiotic susceptibility test results of WPB25

[0042] The results in Table 1 show that, Weissellaparamesenteroides WPB25 showed sensitivity to tetracycline, clindamycin, gentamicin, erythromycin, chloramphenicol, and ampicillin, indicating that the strain has good safety characteristics within the range of antibiotics tested.

[0043] Example 2 contain Weissellaparamesenteroides Preparation of liquid formulation of WPB25: Will Weissellaparamesenteroides After two generations of activation, WPB25 cells were inoculated into MRS liquid medium at a 2% (v / v) inoculum and incubated statically at 37°C for 24 hours. After incubation, the cells were collected by centrifugation at 5000 rpm for 10 minutes at 4°C, washed twice with 0.9% physiological saline, and adjusted using the plate count method. Weissellaparamesenteroides WPB25 has a viable bacterial count of 1×10⁻⁶. 10 CFU / mL, to obtain a concentration of Weissellaparamesenteroides WPB25 liquid bacterial agent.

[0044] Example 3 1. Experimental animals and grouping Twenty-five 7-week-old male C57BL / 6J mice were used. After acclimatizing to standard conditions for one week, they were uniformly fed a high-fat diet (#D12492) for 60 consecutive days. Mice with significant weight gain were selected and randomly divided into a high-fat model group (HFD group) and a WPB25 group, with no significant differences between the groups. Each group consisted of 5 mice. Throughout the feeding period, environmental conditions were uniformly maintained at a temperature of 23℃±1℃, humidity of 50%±5%, and a 12 h / 12 ​​h light-dark cycle. All mice had free access to food and water.

[0045] 2. Feeding method The model group was administered 200 μL of physiological saline per day per mouse by gavage, and the WPB25 group was administered 200 μL of the solution prepared in Example 2 by gavage. Weissellaparamesenteroides WPB25 liquid bacterial agent was administered to mice daily at a fixed time. Food intake and mouse weight were recorded weekly for an 8-week treatment period. At the end of the experiment, mice were starved for 12 hours, weighed, blood was collected from the orbital rim, organs were harvested and weighed, and immediately flash-frozen in liquid nitrogen. After necropsy, the liver, white fat (subcutaneous fat, epididymal fat, perirenal fat), kidneys, pancreas, and spleen were removed, rinsed with physiological saline, blotted dry with filter paper, and weighed.

[0046] 3. Weight changes Mouse weight changes as follows Figure 3 As shown, compared with the HFD group, after... Weissellaparamesenteroides From week 5 to week 8 after WPB25 intervention, the body weight of mice was significantly lower than that of the HFD group, indicating that... Weissellaparamesenteroides WPB25 can alleviate weight gain in mice on a high-fat diet.

[0047] 4. Mouse organ index: The results are as follows Figure 4 As shown, compared with the HFD group, after... Weissellaparamesenteroides WPB25 intervention resulted in decreased liver organ indices, as well as decreased subcutaneous fat, epididymal fat, perirenal fat, and white fat organ indices in mice. These results indicate that... WeissellaparamesenteroidesWPB25 intervention helps alleviate fat and liver organ hypertrophy.

[0048] 5. Liver disease status: Liver tissues from mice in all groups were fixed with 4% paraformaldehyde, followed by standard treatments including dehydration, clearing, paraffin embedding, and sectioning. Sections were stained with hematoxylin and eosin (H&E), and morphological changes were observed under an optical microscope. Liver tissues from mice in the HFD and WPB25 groups were fixed with 4% fixative, dehydrated, cleared, paraffin-embedded, embedded, and sectioned before H&E staining.

[0049] The results are as follows Figure 5 As shown, H&E staining revealed that the HFD group exhibited significant microvesicular steatosis in liver tissue, characterized by increased lipid accumulation, numerous lipid vacuoles, cytoplasmic leakage, cell swelling, and impaired cell membrane integrity. In the WPB25 group, the number of lipid droplets in hepatocytes was significantly reduced after intervention, and the number of lipid vacuoles in hepatocytes was smaller and fewer. Weissellaparamesenteroides WPB25 intervention helps alleviate hepatic lipid degeneration.

[0050] 6. Fatty lesions: Adipocyte diameter is a direct histological indicator of the severity of obesity. Epididymal adipose tissue from mice in all groups was fixed with 4% paraformaldehyde, followed by standard processing including dehydration, clearing, paraffin embedding, and sectioning. Sections were then stained with hematoxylin and eosin (H&E) for observation and analysis.

[0051] The results are as follows Figure 6 As shown, the adipocytes in the HFD group exhibited significant hypertrophy. Weissellaparamesenteroides After WPB25 intervention, the diameter of fat follicles decreased, indicating that... Weissellaparamesenteroides Treatment with WPB25 can effectively reduce the enlargement of white adipocytes induced by HFD.

[0052] 7. Determination of liver biochemical indicators Liver biochemical parameters of mice in the HFD and WPB25 groups were measured using a kit from Jiangsu Edison Biotechnology Co., Ltd. The results are shown in Table 2.

[0053] Table 2- Weissella paramesenteroides Effects of WPB25 on Liver Biochemical Indicators

[0054] As shown in Table 2, compared with the HFD group, the WPB25 group had lower total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels, and significantly higher high-density lipoprotein cholesterol (HDL-C) levels. P <0.05). Leptin (LEP) was significantly reduced (P <0.001), adiponectin (APN / ADP) was significantly increased ( P <0.01), indicating that... Weissella paramesenteroides WPB25 intervention can improve lipid metabolism in obese mice by regulating leptin and adiponectin levels.

[0055] Malondialdehyde (MDA) is the end product of lipid peroxidation and a key biomarker for measuring oxidative stress levels and cellular damage in the body. Compared with the HFD group, after... Weissella paramesenteroides WPB25 intervention reduced MDA content in mouse livers, indicating that... Weissella paramesenteroides WPB25 intervention helps prevent lipid peroxidation. Furthermore, compared to the HFD group, the WPB25 group showed significantly lower levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). P <0.01), the anti-inflammatory factor interleukin-10 (IL-10) was significantly elevated ( P <0.01). This indicates Weissella paramesenteroides WPB25 helps regulate immunity and suppress inflammatory responses.

[0056] 8. Determination of serum biochemical indicators Serum biochemical parameters of mice were determined using a kit from Jiangsu Edison Biotechnology Co., Ltd. The results are shown in Table 3. Table 3- Weissella paramesenteroides Effects of WPB25 on serum biochemical parameters

[0057] As shown in Table 3, compared with the HFD group, the WPB25 group had significantly lower leptin levels and significantly higher adiponectin levels. P <0.001), indicating Weissella paramesenteroides WPB25 can regulate lipid metabolism; the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were reduced in mice in the WPB25 group, indicating that... Weissella paramesenteroides WPB25 intervention alleviated liver damage caused by a high-fat diet in obese mice and had a protective effect on the liver. Furthermore, the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) levels, and superoxide dismutase (SOD) levels were significantly increased in the WPB25 group mice. P <0.05); malondialdehyde (MDA) content was significantly reduced ( P <0.01), indicating that Weissella paramesenteroides WPB25 can enhance the antioxidant capacity of mice on a high-fat diet by reducing MDA and increasing SOD.

[0058] Furthermore, compared with the HFD group, the WPB25 group showed decreased levels of the pro-inflammatory factor TNF-α, and significantly decreased levels of IL-1β and IL-6. P <0.05); the anti-inflammatory factor IL-10 was significantly elevated ( P <0.05), indicating Weissella paramesenteroides WPB25 can inhibit the inflammatory response in obese mice induced by a high-fat diet.

[0059] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A single plant Weissella paramesenteroides WPB25, characterized in that, The Weissella paramesenteroides WPB25 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37194, on December 25, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. As described in claim 1 Weissella paramesenteroides WPB25, characterized in that, The Weissella paramesenteroides The 16S rDNA sequence of WPB25 is shown in SEQ ID NO.

1.

3. A device comprising the following as described in claim 1 Weissella paramesenteroides The formulation of WPB25.

4. The formulation according to claim 3, characterized in that, The bacterial agent can be a liquid or solid preparation.

5. The formulation as described in claim 4, characterized in that, In the liquid formulation Weissella paramesenteroides The content of WPB25 is 1.0 × 10⁻⁶. 7 ~1.0×10 10 CFU / mL; in the solid dosage form Weissella paramesenteroides The content of WPB25 is 1.0 × 10⁻⁶. 7 ~1.0×10 10 CFU / g.

6. The formulation as described in claim 5, characterized in that, The preparation method of the liquid formulation is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Weissella paramesenteroides After two generations of activation, WPB25 cells were inoculated into MRS liquid medium at a volume ratio of 2% and incubated statically at 37°C for 24 hours. After incubation, the cells were collected by centrifugation at 5000 rpm for 10 minutes at 4°C, washed twice with 0.9% physiological saline, and adjusted using the plate count method. Weissella paramesenteroides WPB25 has a viable bacterial count of 1×10⁻⁶. 10 CFU / mL, to obtain a concentration of Weissella paramesenteroides WPB25 liquid bacterial agent.

7. The formulation of claim 6, characterized in that, The MRS liquid culture medium consists of the following components: 10.0 g peptone, 1.0 mL Tween 80, 4.0 g yeast extract, 2.0 g triammonium citrate, 0.05 g manganese sulfate, 5.0 g beef meal, 2.0 g dipotassium hydrogen phosphate, 20.0 g glucose, 0.2 g magnesium sulfate, and 5.0 g sodium acetate, dissolved in 1000 mL distilled water, pH = 6.2 ± 0.2; autoclaved at 121 °C for 15 min.

8. A device as described in claim 1 Weissella paramesenteroides Application of WPB25 in the preparation of products for the prevention, relief or treatment of obesity.

9. A device as described in claim 1 Weissella paramesenteroides Application of WPB25 in the preparation of products for the prevention, relief or treatment of metabolic syndrome.

10. A device as described in claim 1 Weissella paramesenteroides Application of WPB25 in the preparation of products that alleviate inflammation and enhance antioxidant properties.

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