Propionibacterium freudenreichii subsp. shermanii pf310 for relieving obesity by regulating energy metabolism related hormones and application thereof

Abstract

The present application relates to the field of microbial technology and the field of medicine and health, and specifically discloses a propionibacterium freudenreichii subsp. shermanii PF310 for relieving obesity by regulating energy metabolism related hormones and application thereof. Propionibacterium freudenreichii subsp. shermanii The propionibacterium freudenreichii subsp. shermanii (P. shermanii) PF310 is isolated from yak milk in Naqu City, and the preservation number is CGMCC No. 31417. Animal experiments prove that the P. shermanii PF310 provided in the present application realizes a significant anti-obesity effect through a unique energy metabolism related hormone multi-target synergistic regulation mechanism, has the outstanding advantages of strong strain specificity, still effective after heat inactivation, wide application prospect and the like, and provides a new probiotic resource and intervention strategy for the prevention and treatment of obesity.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and pharmaceutical health, specifically relating to a strain of Propionibacterium fischeri subsp. Scheres PF310 and its application in the preparation of products for regulating hormones related to energy metabolism and for the prevention and / or relief of obesity. Background Technology

[0002] Obesity is a major global health problem, closely associated with various metabolic diseases such as cardiovascular disease, type 2 diabetes, and non-alcoholic fatty liver disease. Obesity results from the combined effects of multiple factors, including excessive food intake, insufficient energy expenditure, genetic factors, and environmental factors. In recent years, numerous studies have demonstrated the crucial role of gut microbiota in the development and progression of obesity, making the intervention of obesity through gut microbiota regulation a new research hotspot.

[0003] Probiotics, as a class of live microorganisms beneficial to the host's health, show promising applications in promoting weight loss by improving gut microbiota structure and regulating metabolism and immune function. Currently reported probiotics with anti-obesity potential mainly include Lactobacillus, Bifidobacterium, and Akkermansia. However, current research on the anti-obesity mechanisms of probiotics mainly focuses on directly regulating adipose tissue metabolism, such as inhibiting adipocyte differentiation (transcription factors like PPARγ and C / EBPα), downregulating the expression of lipases (FAS, ACC, SCD-1, etc.), and promoting fat breakdown. Research on their anti-obesity effects through the regulation of energy metabolism-related hormones remains lacking.

[0004] Hormones related to energy metabolism are key molecules regulating appetite, energy intake, and expenditure. Among them, glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by intestinal L cells, which delays gastric emptying, suppresses appetite, and promotes insulin secretion; leptin, mainly secreted by adipocytes, transmits energy reserve signals to the central nervous system, suppressing appetite and promoting energy expenditure; ghrelin, mainly secreted by the stomach, is the only known peripheral appetite-stimulating hormone, with elevated levels during fasting to promote food intake; and insulin, secreted by pancreatic β cells, regulates blood glucose homeostasis, and obese individuals often have hyperinsulinemia and insulin resistance. These four hormones constitute a sophisticated appetite-energy metabolism regulatory network, and its imbalance is an important pathological basis for obesity. In recent years, drugs targeting these hormones (such as the GLP-1 receptor agonist smegglutide) have made breakthrough progress in clinical weight loss treatment, but they have problems such as injection administration and gastrointestinal side effects.

[0005] Therefore, developing probiotic strains that can safely and effectively regulate endogenous energy metabolism-related hormones via oral administration is of significant scientific importance and clinical application value. Summary of the Invention

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of Propionibacterium fischeri subsp. Scheres (… Propionibacterium freudenreichii subsp. shermanii PF310, the preservation number of this strain is CGMCC No. 31417.

[0008] The *Propionibacterium Fischeriensis* subsp. *Schärni* PF310 strain was isolated from yak milk samples. Morphological observation, Gram staining identification, and 16S rRNA gene sequence analysis confirmed it to be *Propionibacterium Fischeriensis* subsp. *Schärni*. This strain was deposited on July 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Experiments have verified that *Propionibacterium freundii* subsp. *Schärni* PF310 possesses unique metabolic regulatory functions, significantly modulating the levels of energy metabolism-related hormones in mammals. Specifically, this strain upregulates the levels of glucagon-like peptide-1 (GLP-1) and leptin, while downregulating the levels of ghrelin and insulin, forming a multi-hormonal synergistic regulatory effect. This multi-target synergistic regulatory mechanism enables the PF310 strain to exert a comprehensive anti-obesity effect from multiple dimensions, including appetite suppression, increased energy expenditure, and improved insulin sensitivity.

[0010] Furthermore, experiments confirmed that *Propionibacterium freundii* subsp. *Schärni* PF310 significantly reduced serum triglyceride and total cholesterol levels in mammals, improved insulin resistance, and effectively prevented and / or alleviated obesity. This strain exhibited significant strain-specific function; the control strain PF37, also a *Propionibacterium freundii* subsp. *Schärni*, did not possess these functions.

[0011] In a second aspect, the present invention provides a probiotic composition comprising Propionibacterium fischeri subsp. Scheres PF310 as described in the first aspect of the present invention, and a pharmaceutically, food-grade, or feed-grade acceptable carrier.

[0012] The carrier can be selected according to the intended use and formulation of the composition. For example, pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, etc.; food-acceptable carriers include, but are not limited to, water, dairy products, fruit juices, cereal flours, sugars, starches, etc.; and feed-acceptable carriers include, but are not limited to, corn flour, soybean meal, wheat bran, dicalcium phosphate, etc.

[0013] In this invention, the Propionibacterium fischeri subsp. Scheres PF310 can exist in the composition in live form, inactivated form, or as a component of the bacterial cell.

[0014] In this invention, the term "live bacterial form" refers to the form of microbial cells possessing metabolic activity and reproductive capacity. The term "inactivated form" refers to the form of microbial cells that have lost metabolic activity and reproductive capacity but retain their cell structure after being treated by physical or chemical methods, including but not limited to heat inactivation, irradiation inactivation, and chemical inactivation. The term "cell component form" refers to cell components extracted or prepared from microbial cells, including but not limited to cell lysates, cell wall components, cell membrane components, and intracellular extracts.

[0015] Based on the different forms described above, the compositions of the present invention can be prepared into various formulation types. Specifically, the compositions can be live bacterial formulations, i.e., products containing active PF310 strains; they can be metabiotic formulations, i.e., products containing PF310 cell components and / or its metabolites; or they can be fermentation product formulations, i.e., products containing PF310 fermentation products (including cell components and fermentation metabolites). These formulation forms can be selected and prepared according to actual application requirements.

[0016] Preferably, the viable count of *Propionibacterium freundii* subsp. *Schärni* PF310 in the live bacterial preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, or the number of bacterial cells used in the preparation of postbiotic formulations is not less than 1×10⁻⁶. 9 per g.

[0017] Thirdly, the present invention provides the use of Propionibacterium fischeri subsp. Scheres PF310 as described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, in the preparation of products for regulating hormones related to energy metabolism in mammals.

[0018] The energy metabolism-related hormones include, but are not limited to, glucagon-like peptide-1 (GLP-1), leptin, ghrelin, and insulin. The regulation includes one or more of the following mechanisms: upregulating GLP-1 levels, upregulating leptin levels, downregulating ghrelin levels, and downregulating insulin levels.

[0019] Furthermore, the regulation includes simultaneously upregulating GLP-1 and leptin levels, and simultaneously downregulating ghrelin and insulin levels, forming a multi-hormonal synergistic regulatory effect. This multi-hormonal synergistic regulatory effect enables PF310 to comprehensively regulate energy metabolism through multiple physiological pathways: by upregulating GLP-1 to delay gastric emptying, suppress appetite, and promote insulin secretion; by upregulating leptin to enhance central satiety signals and promote energy consumption; by downregulating ghrelin to reduce the urge to eat; and by downregulating insulin to improve insulin resistance.

[0020] In one specific embodiment of the present invention, experimental data showed that, compared with the high-fat model group, the serum GLP-1 level of mice in the PF310 intervention group was significantly increased by 57.16%, indicating that PF310 can effectively promote the secretion of GLP-1 by intestinal L cells, exerting physiological effects of suppressing appetite, delaying gastric emptying, and promoting insulin secretion; the leptin level was significantly increased by 51.74%, indicating that PF310 can effectively improve leptin resistance commonly seen in obesity, enhance central satiety signals, and promote energy consumption; the ghrelin level was significantly decreased by 41.26%, indicating that PF310 can effectively inhibit the secretion of orexin and reduce the urge to eat; and the insulin level was significantly decreased by 31.76%, indicating that PF310 can effectively improve hyperinsulinemia and insulin resistance.

[0021] Fourthly, the present invention provides the use of Propionibacterium fischeri subsp. Scheres PF310 as described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, in the preparation of products for the prevention and / or relief of obesity.

[0022] In one specific embodiment of the present invention, experimental data showed that in a high-fat diet-induced obese mouse model, intervention with the PF310 strain significantly inhibited mouse weight gain, with an increase of only 1.20%, far lower than the 18.25% in the high-fat model group, and the effect was superior to the positive drug orlistat group (5.05%). Heat-inactivated PF310 cells also showed a significant inhibitory effect on weight gain (4.74%), indicating that the effect of this strain is independent of the cell's active state.

[0023] Fifthly, the present invention provides the use of Propionibacterium fischeri subsp. Scheres PF310 as described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, in the preparation of products for lowering triglycerides and / or total cholesterol.

[0024] In one specific embodiment of the present invention, experimental data showed that the serum triglyceride level of mice in the PF310 intervention group was significantly reduced by 20.02%, and the total cholesterol level was significantly reduced by 26.20%; the heat-inactivated PF310 group also showed a significant lipid-lowering effect, with triglycerides reduced by 23.39% and total cholesterol reduced by 26.84%. This indicates that PF310 can effectively improve obesity-related lipid metabolism disorders.

[0025] In a sixth aspect, the present invention provides the use of Propionibacterium fischeri subsp. Scheres PF310 as described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, in the preparation of a product for improving insulin resistance.

[0026] In one specific embodiment of the present invention, experimental data showed that the serum insulin level of mice in the PF310 intervention group was significantly reduced by 31.76%, indicating that this strain can effectively improve hyperinsulinemia and insulin resistance. The heat-inactivated PF310 group also showed a significant reduction in insulin levels (31.37%), further confirming the metabolic regulatory function of this strain.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention is the first to discover and confirm that *Propionibacterium freundii* subsp. *Schärni* PF310 can significantly regulate the levels of multiple key energy metabolism-related hormones in mammals, forming a complete appetite-energy metabolism regulatory network. Experiments have shown that PF310 can simultaneously upregulate GLP-1 and leptin levels, and simultaneously downregulate ghrelin and insulin levels, forming a multi-hormonal synergistic regulatory effect. This synergistic regulatory effect of the aforementioned four hormones has never been reported in the prior art.

[0029] This invention also confirms that heat-inactivated PF310 cells can still significantly regulate the levels of hormones related to energy metabolism. This characteristic indicates that the effect of PF310 does not depend on the active state of the cells, and its cell components or metabolites are the main effector substances. Heat-inactivated preparations have higher safety, stability and feasibility for standardized production, can be used in people with weakened immune systems, do not require cold chain transportation and storage, and have a wider range of applications.

[0030] Furthermore, this invention demonstrates, through comparison with the positive control drug orlistat, that PF310's regulation of the aforementioned hormones is a direct effect rather than a secondary effect of weight loss.

[0031] Furthermore, this invention also demonstrates through experiments with the control strain PF37 that the function of PF310 exhibits significant strain specificity. The PF37 intervention group, also belonging to *Propionibacterium freundii* subsp. *Schärne*, failed to significantly increase GLP-1 and leptin levels, failed to significantly decrease ghrelin and insulin levels, and failed to significantly inhibit weight gain or reduce blood lipids. This control experiment directly proves that the aforementioned functions of PF310 are not universal properties of *Propionibacterium freundii* subsp. *Schärne*, but rather unique and unpredictable technical effects specific to the PF310 strain.

[0032] This invention also achieved significant anti-obesity effects. In a high-fat diet-induced obese mouse model, the PF310 intervention group showed a weight gain of only 1.20% over 6 weeks, significantly lower than the 18.25% in the high-fat model group and also superior to the 5.05% in the positive control drug orlistat group; the heat-inactivated PF310 group showed a weight gain of 4.74%, also significantly lower than the high-fat model group. Furthermore, the PF310 intervention group showed a significant reduction of 20.02% in serum triglyceride levels and 26.20% in total cholesterol levels, indicating that PF310 can effectively improve obesity-related lipid metabolism disorders.

[0033] In summary, the Propionibacterium fischeri subsp. Scheres PF310 provided by this invention achieves significant anti-obesity effects through a unique multi-target synergistic regulatory mechanism of energy metabolism-related hormones. It has outstanding advantages such as strong strain specificity, effectiveness even after heat inactivation, and broad application prospects. It can be used as a candidate for functional food and drug development or as a postbiotic preparation, and can be widely applied in various food carriers such as dairy products, beverages, and baked goods, providing a novel probiotic resource and intervention strategy for the prevention and treatment of obesity. Attached Figure Description

[0034] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0035] Figure 1 This is a colony morphology diagram of Propionibacterium fischeri subsp. Schaeferi PF310.

[0036] Figure 2 Gram-stained microscopic image of Propionibacterium fischeri subsp. Scheres PF310.

[0037] Figure 3 The image shows the results of oil red O staining to detect lipid accumulation in Caenorhabditis elegans.

[0038] Figure 4The following are the results of transcriptome analysis of *C. elegans*. Figures A and B are differential gene volcano plots, where each dot represents a gene. Red dots indicate genes significantly upregulated compared to the control group, blue dots indicate genes significantly downregulated compared to the control group, and gray dots indicate genes with no significant difference compared to the control group. Figures C and D are KEGG pathway enrichment analysis results, with the horizontal axis representing the significance of enrichment and the vertical axis representing the enriched KEGG pathway. Figures E and F are GO function enrichment analysis results, with the horizontal axis representing the significance of enrichment and the vertical axis representing the name of the enriched GO function.

[0039] Figure 5 The graphs show the changes in mouse body weight. A is a curve showing the changes in body weight of each group of mice during the 6-week intervention period; B is a bar chart showing the percentage increase in body weight of each group of mice during the 6-week intervention period. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0040] Figure 6 A comparative graph showing the growth curves of 11 strains of Propionibacterium fischeri subsp. Scheres at a fermentation temperature of 37℃. Detailed Implementation

[0041] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0042] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0043] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] The control strain used in the following examples, Propionibacterium fischeri subsp. Scheres PF37, is described and disclosed in patent document CN120173826B.

[0047] Example 1: Isolation and identification of Propionibacterium fischeri subsp. Schaeferi PF310

[0048] 1. Culture medium preparation

[0049] This invention uses YEL medium to culture Propionibacterium fischeri subsp. Scheres PF310, the composition of which is: 21.6 g / L 60% lactic acid solution, 10 g / L yeast extract, 10 g / L peptone, 0.328 g / L dipotassium hydrogen phosphate, and 0.056 g / L manganese sulfate. The solid medium is prepared by adding 15 g / L agar to the above formula.

[0050] 2. Strains Isolation

[0051] Yak milk was collected from Nagqu City and inoculated into liquid YEL medium. The culture was enriched under anaerobic conditions at 37°C for 72 hours. The enriched culture was then serially diluted and plated onto YEL agar plates, and incubated anaerobically at 37°C for 72 to 96 hours. After colony growth, single colonies were selected based on colony morphology (size, color, edge, gloss, etc.) and purified by streaking three times on YEL agar plates to obtain pure cultures. The purified strains were then inoculated into liquid YEL medium for further cultivation.

[0052] 3. Strain identification

[0053] Morphological identification: The isolated and purified strain was inoculated onto YEL agar plates and anaerobically cultured at 30°C for 96 hours. Colony morphology was then observed. Results showed that the strain formed round, smooth, raised, well-defined, opaque white colonies on YEL agar plates. Figure 1 The fresh culture was Gram-stained, and the bacterial morphology was observed under a light microscope. The results showed that the strain was Gram-positive, with short, granular cells arranged singly or in groups, and no spores. Figure 2 ).

[0054] Sequencing identification: Using bacterial culture medium as a template, PCR amplification was performed using the universal primers 16S rRNA 27F / 1492R. The PCR amplification products were then sent to a sequencing company for sequencing. The sequencing sequence is shown in SEQ ID No. 1. After comparing the sequencing results with the NCBI database, the strain was identified as *Propionibacterium fischeri* subsp. *shesleyanum* (…). Propionibacterium freudenreichii sub sp.shermanii The strain was named *Propionibacterium fischeri* subsp. *Schärni* PF310. Propionibacterium freudenreichii sub sp.shermanii PF310).

[0055] 4. Preservation of microbial strains

[0056] The aforementioned *Propionibacterium freundii* subsp. *Schär* PF310 was deposited on July 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The accession number is CGMCC No. 31417, and the classification name is *Propionibacterium freundii* subsp. *Schär* (…). Propionibacterium freudenreichii subsp. shermanii ).

[0057] Example 2: Propionibacterium fischeri subsp. Scheres PF310 reduces lipid accumulation in Caenorhabditis elegans.

[0058] Caenorhabditis elegans strain N2 was cultured on high-glucose NGM agar plates coated with Escherichia coli OP50. The composition of the high-glucose NGM agar plates was: tryptone 2.5 g / L, sodium chloride 3 g / L, glucose 1.8 g / L, agar 22 g / L, potassium dihydrogen phosphate 2.7 g / L, dipotassium hydrogen phosphate 0.9 g / L, calcium chloride 1 mmol / L, magnesium sulfate 1 mmol / L, calcium chloride 5 mg / L, and cholesterol 5 mg / L. The rearing temperature was 20℃. Escherichia coli OP50 was cultured on LB medium at 37℃. The M9 buffer for rewashing and resuspending the nematodes was formulated as follows: potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 7.35 g / L, sodium chloride 5 g / L, and magnesium sulfate 0.12 g / L.

[0059] L1 stage Caenorhabditis elegans were inoculated onto plates containing different foods and cultured (3 biological replicates per group):

[0060] Control group: Escherichia coli OP50 was used as food. The preparation method was as follows: 100 μL of overnight cultured Escherichia coli OP50 was added to a high-glucose NGM agar plate, spread evenly, and incubated overnight at 37°C before use.

[0061] PF310 group: Escherichia coli OP50 supplemented with Propionibacterium freundii subsp. serum PF310 was used as food. Preparation method: 1 mL of Propionibacterium freundii subsp. serum PF310 bacterial suspension with OD600=3 was taken, inactivated at 100℃ for 30 minutes, centrifuged, and the supernatant was removed. The suspension was resuspended in 100 μL of overnight cultured Escherichia coli OP50 bacterial suspension, then added dropwise to a high-glucose NGM agar plate, spread evenly, and incubated overnight at 37℃ before use.

[0062] Group PF37: Escherichia coli OP50 supplemented with Propionibacterium freundii subsp. Scheresii PF37 was used as food. Preparation method: 1 mL of Propionibacterium freundii subsp. Scheresii PF37 bacterial suspension with OD600=3 was taken, inactivated at 100℃ for 30 minutes, centrifuged, and the supernatant was removed. The suspension was resuspended in 100 μL of overnight cultured Escherichia coli OP50, then added dropwise to a high-glucose NGM agar plate, spread evenly, and incubated overnight at 37℃ before use.

[0063] Positive drug group: Escherichia coli OP50 containing 9 μg / mL orlistat was used as food. The preparation method was as follows: 100 μL of overnight cultured Escherichia coli OP50 containing 9 μg / mL orlistat was added to a high-glucose NGM agar plate, spread evenly, and incubated overnight at 37°C before use.

[0064] Caenorhabditis elegans inoculated onto plates were cultured at 20°C for 2 days and then collected into EP tubes. The tubes were stained with Oil Red O staining kit, and the nematodes were observed and photographed under an upright microscope after staining.

[0065] Oil Red O staining results showed ( Figure 3 In the control group, a large number of lipid droplets stained red were observed in the nematodes, mainly distributed in the intestine and subcutaneous tissue, indicating that lipid accumulation in the nematodes was significant under high sugar culture conditions.

[0066] Compared with the control group, the number of red lipid droplets in the PF310 group was significantly reduced and the staining intensity was significantly decreased, indicating that the inactivated Propionibacterium fischeri subsp. Scheres PF310 can effectively reduce lipid accumulation in Caenorhabditis elegans.

[0067] Oil Red O staining results of nematodes in the control strain PF37 group showed no significant difference from those in the control group. The number of lipid droplets and staining intensity were not significantly reduced, indicating that the PF37 strain, which is also a subspecies of Propionibacterium fischeri, does not have the function of reducing lipid accumulation, thus verifying the strain specificity of PF310 function.

[0068] The lipid accumulation in the positive drug orlistat group of nematodes was also significantly reduced, with an effect comparable to that of the PF310 group, indicating that orlistat, as a lipase inhibitor, can effectively exert a lipid-lowering effect in this experimental system.

[0069] The results of this embodiment show that *Propionibacterium freundii* subsp. *Schärni* PF310 can significantly reduce lipid accumulation in *C. elegans*, while the control strain PF37 has no such effect. This demonstrates that PF310 has a function in regulating lipid metabolism, and also confirms that this function is strain-specific and not a universal property of *Propionibacterium freundii* subsp. *Schärni*.

[0070] Example 3: Propionibacterium fischeri subsp. Scheres PF310 reduces fatty acid content in Caenorhabditis elegans.

[0071] The *C. elegans* worms from Example 2 were collected, resuspended in 1 mL of M9 buffer, and 900 μL of worms were used for GC-MS analysis to determine the fatty acid content. The remaining 100 μL was used to determine the total protein content using a BCA protein quantification kit.

[0072] Each group had three biological replicates, and each sample had three technical replicates. Results are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0073] As shown in Table 1, compared with the control group, feeding nematodes with inactivated Propionibacterium freundii subsp. serratum PF310 significantly reduced the levels of 20 fatty acids. While the levels of the same 20 fatty acids in the control strain Propionibacterium freundii subsp. serratum PF37 showed a slight decreasing trend, none of them reached statistical significance compared to the control group, indicating that PF37 does not possess lipid regulatory functions comparable to PF310.

[0074] Table 1. Fatty acid content in *C. elegans* (mg / g protein)

[0075]

[0076] Note: Data in the table are expressed as mean ± standard deviation, and * indicates a significant difference compared to the control group.

[0077] Example 4: Regulating one-carbon and lipid metabolism in *Propionibacterium fischeri* subsp. *Schärni* using PF310.

[0078] RNA was extracted from *C. elegans* in Example 2 for transcriptome analysis. The experimental results are as follows: Figure 4 As shown.

[0079] 1. Differentially expressed gene analysis

[0080] Figure 4 A shows a volcano plot of differentially expressed genes between the PF310 group and the control group. Each dot in the plot represents a gene; red dots indicate significantly upregulated genes, blue dots indicate significantly downregulated genes, and gray dots indicate genes with no significant difference. The results showed that, compared with the control group, the PF310 group had 156 significantly upregulated genes and 1025 significantly downregulated genes.

[0081] Figure 4 B shows a volcano plot of differentially expressed genes between the PF37 group and the control group. The results show that, compared with the control group, only 35 genes were significantly upregulated and 100 genes were significantly downregulated in the PF37 group, and the changes in gene expression profile were much smaller than those in the PF310 group.

[0082] This result indicates that PF310 intervention has a broad and significant effect on gene expression in Caenorhabditis elegans, while the effect on the control strain PF37 is very limited, further confirming the strain-specificity of PF310 function.

[0083] 2. KEGG pathway enrichment analysis

[0084] Figure 4 Figure C shows the results of KEGG pathway enrichment analysis of differentially expressed genes between the PF310 group and the control group. The horizontal axis represents the significance of enrichment, and the vertical axis represents the names of the enriched KEGG pathways. The results show that the pathways significantly enriched by PF310 intervention include: one-carbon metabolism, amino acid metabolism-related pathways, and energy metabolism-related pathways.

[0085] Figure 4 D presents the results of KEGG pathway enrichment analysis of differentially expressed genes between the PF37 group and the control group. The results showed that the main enriched pathways after PF37 intervention included lysosomes, drug metabolism-cytochrome P450, and exogenous substance metabolism-cytochrome P450. Pathways related to lipid metabolism or energy metabolism did not show significant enrichment. This result indicates that the mechanisms of action of PF37 and PF310 are fundamentally different.

[0086] 3. GO Functional Enrichment Analysis

[0087] Figure 4 Figure E shows the results of GO functional enrichment analysis of differentially expressed genes (biological process level) between the PF310 group and the control group. The horizontal axis represents the significance of enrichment, and the vertical axis represents the names of the enriched GO functions. The results show that PF310 intervention significantly enriched multiple functional genes related to lipid metabolism, indicating that PF310 has a broad regulatory role in lipid metabolism.

[0088] Figure 4 F shows the results of GO functional enrichment analysis of differentially expressed genes between the PF37 group and the control group. The results show that after PF37 intervention, few functional genes related to lipid metabolism were enriched, further confirming that the regulatory effect of PF310 on lipid metabolism is strain-specific and not a universal property of Propionibacterium fischeri subsp. Sheehaniae.

[0089] This comparison further demonstrates that the regulatory effect of PF310 on lipid metabolism is its unique function, while PF37 does not have the same effect.

[0090] The results of this embodiment indicate that Propionibacterium fischeri subsp. Schaeferi PF310 mainly reduces lipid accumulation by altering lipid metabolism-related pathways in Caenorhabditis elegans.

[0091] Example 5: Propionibacterium fischeri subsp. Scheres PF310 reduces weight gain in obese mice.

[0092] Forty-eight 6-week-old SPF-grade male C57 mice were used in this experiment. After one week of acclimatization, eight mice were randomly selected as the control group and fed a normal diet with an energy ratio of 22.47% protein, 12.11% fat, and 65.42% carbohydrates. The remaining 42 mice were fed a high-fat diet to model obesity. The high-fat diet was the D12492 diet, with an energy ratio of 20% protein, 60% fat, and 20% carbohydrates.

[0093] Modeling was maintained for 8 weeks. After modeling, the obese modeling mice fed a high-fat diet were randomly divided into 5 groups of 8 mice each. They continued to be fed a high-fat diet and underwent gavage intervention according to the grouping shown in Table 2. The intervention period was 6 weeks.

[0094] Table 2. Mouse grouping and gavage intervention

[0095]

[0096] The total food consumption of mice during the 6-week intervention was recorded. Divided by the number of mice and the total number of intervention days, the daily food intake per mouse was calculated (see Table 3). The average food intake of mice in the PF310 and HK_PF310 groups decreased significantly. The average food intake of mice in the PF37 group did not decrease significantly.

[0097] Table 3 Average food intake of mice

[0098]

[0099] # indicates that there is a significant difference between the test results of this group and the test results of the high-fat group. The calculation method is one-way ANOVA.

[0100] During the intervention period, the weight of mice in each group was measured and recorded at fixed times each week, and the percentage of weight gain was calculated. The results are as follows: Figure 5 As shown.

[0101] Figure 5 A shows the body weight change curves of mice in each group during the 6-week intervention period. Results showed that the body weight of mice in the control group remained relatively stable throughout the intervention period, with a slight but minimal increase; the body weight of mice in the high-fat group showed a continuous upward trend, gradually increasing from week 1 of the intervention; the body weight change curve of the PF37 group was similar to that of the high-fat group, with a gradual increase in body weight; the body weight of mice in the PF310 and HK_PF310 groups did not increase significantly throughout the intervention period, and the curves tended to flatten; the body weight increase of mice in the positive control group was between that of the high-fat and PF310 groups. By week 6, the endpoint of the intervention, the average body weight of mice in the PF310 and HK_PF310 groups was significantly lower than that of the high-fat group, while the average body weight of mice in the PF37 group was not significantly different from that of the high-fat group.

[0102] Figure 5B shows the percentage weight gain of each group of mice during the 6-week intervention period. The results showed that the average weight gain of mice in the high-fat group was 18.25%, and the average weight gain of mice given Propionibacterium Fischeriensis subsp. Sheae PF37 via gavage was 11.81%. However, the percentage weight gain of mice given Propionibacterium Fischeriensis subsp. Sheae PF310 via gavage and inactivated Propionibacterium Fischeriensis subsp. Sheae PF310 via gavage were 1.20% and 4.74%, respectively, which were significantly lower than those in the high-fat group and also lower than the 5.05% in the positive control group.

[0103] The results of this embodiment show that:

[0104] PF310 significantly inhibited weight gain induced by a high-fat diet. The weight gain rate of mice in the PF310 intervention group was only 1.20% over 6 weeks, far lower than the 18.25% in the high-fat model group, demonstrating excellent anti-obesity effects.

[0105] Heat-inactivated PF310 cells still retain their anti-obesity function. The weight gain rate of mice in the HK_PF310 group was 4.74%, which was also significantly lower than that in the high-fat model group, indicating that the effect of PF310 does not depend on the activity state of the cells, and its cell components are the main effector substances. This characteristic makes PF310 a potential candidate for developing postbiotic formulations with higher safety and better stability.

[0106] The function of PF310 is strain-specific. The intervention group of PF37, which is also Propionibacterium fischeri subsp. Sheehaniae, had no significant effect on body weight, proving that the anti-obesity function of PF310 is a unique technical effect of this strain.

[0107] PF310 was more effective than the positive control drug orlistat. The weight gain rate in the PF310 group was lower than that in the orlistat group, showing superior weight loss results, and without the gastrointestinal side effects that orlistat may cause.

[0108] Example 6: Propionibacterium fischeri subsp. Scheres PF310 reduces triglyceride and total cholesterol levels.

[0109] Serum from the mice in Example 5 was used to determine the levels of serum triglycerides (TG) and total cholesterol (TCHO) using a kit. The results are shown in Table 4.

[0110] Table 4 Serum TG and TCHO levels in mice

[0111]

[0112] # indicates that there is a significant difference between the test results of this group and the test results of the high-fat group. The calculation method is one-way ANOVA.

[0113] Triglyceride levels in mice were significantly higher in the high-fat diet group than in the control group, indicating that the high-fat diet successfully induced lipid metabolism disorder. Compared with the high-fat diet group, mice administered *Propionibacterium freundii* subsp. *S. sherry* PF310 via gavage showed a significant 20.02% reduction in serum triglycerides (TG), while mice administered inactivated *Propionibacterium freundii* subsp. *S. sherry* PF310 via gavage showed a significant 23.39% reduction in serum TG, with the lipid-lowering effect slightly better than the live bacteria group. Orlistat, a positive control, significantly reduced serum TG in mice by 21.74%, indicating that orlistat was effective as a positive control. However, gavage administration of *Propionibacterium freundii* subsp. *S. sherry* PF37 via gavage had no significant effect on serum TG levels in mice.

[0114] Total cholesterol levels were significantly higher in the high-fat diet group than in the control group, indicating that a high-fat diet leads to severe cholesterol metabolism disorders. Compared with the high-fat diet group, mice administered *Propionibacterium freundii* subsp. *S. sherry* PF310 via gavage showed a significant decrease in serum TCHO (26.20%), and mice administered inactivated *Propionibacterium freundii* subsp. *S. sherry* PF310 via gavage showed a significant decrease in serum TCHO (26.84%). There was no significant difference in serum TCHO levels between the high-fat diet group and the high-fat diet group when administered *Propionibacterium freundii* subsp. *S. sherry* PF37 via gavage. The positive control group showed no significant difference in serum TCHO levels compared to the high-fat diet group, indicating that orlistat primarily affects triglyceride metabolism and has limited regulatory effect on total cholesterol.

[0115] The results of this embodiment show that:

[0116] PF310 significantly reduced serum triglyceride levels, with effects comparable to or even better than the positive control drug orlistat. Heat-inactivated bacteria showed slightly better results than live bacteria, suggesting that the lipid-lowering effect of PF310 may primarily originate from its bacterial components. Furthermore, PF310 exhibits unique advantages in regulating cholesterol metabolism.

[0117] Example 7: Propionibacterium fischeri subsp. Scheres PF310 reduced serum insulin and ghrelin levels in mice and promoted the secretion of leptin and GLP-1 hormones.

[0118] Serum from mice in Example 5 was used to measure the levels of four hormones: insulin (INS), leptin (LEP), glucagon-like peptide-1 (GLP-1), and ghrelin. The results are shown in Table 5.

[0119] Table 5. Serum vitamin INS, LEP, GLP-1, and Ghrelin levels in mice.

[0120]

[0121] # indicates that there is a significant difference between the test results of this group and the test results of the high-fat group. The calculation method is one-way ANOVA.

[0122] Insulin levels were elevated in high-fat mice, indicating that the high-fat diet led to significant insulin resistance. Compared to the high-fat group, oral administration of *Propionibacterium freundii* subsp. *S. shcherei* PF310 and inactivated *Propionibacterium freundii* subsp. *S. shcherei* PF310 bacterial suspensions resulted in a 31.76% and 31.37% decrease in serum insulin levels, respectively, indicating that PF310 strain intervention reduced insulin resistance in obese mice. The control strain PF37 group and the positive control group showed no significant decrease in insulin levels, suggesting that PF37, also belonging to *Propionibacterium freundii* subsp. *S. shcherei*, does not possess the same function, and that orlistat, as a lipase inhibitor, has limited effect on improving insulin resistance.

[0123] Leptin levels were significantly elevated in mice administered Propionibacterium freundii subsp. serum PF310 via gavage compared to the high-fat group, increasing by 51.74%. Leptin is a hormone secreted by adipocytes that suppresses appetite and promotes energy expenditure. PF310 significantly upregulated leptin levels, indicating its ability to effectively improve leptin resistance commonly seen in obesity and enhance central satiety signals. Intervention with inactivated Propionibacterium freundii subsp. serum PF310 also increased LEP levels by 23.98%. The control strain PF37 and the positive control drug had little effect on LEP levels.

[0124] Glucagon-like peptide-1 (GLP-1) levels were detected in mice in the high-fat diet group, showing that serum GLP-1 levels were significantly lower than those in the control group, indicating that a high-fat diet inhibited GLP-1 secretion. Compared with the high-fat diet group, gavage administration of *Propionibacterium freundii* subsp. *shelenia* PF310 bacterial suspension and inactivated *Propionibacterium freundii* subsp. *shelenia* PF310 bacterial suspension significantly increased GLP-1 levels by 57.16% and 45.01%, respectively, both exceeding the effects of the control strain PF37 and the positive control drug. GLP-1 is an incretin hormone synthesized by intestinal cells; increasing GLP-1 secretion can delay gastric emptying, reduce food intake, and lower blood glucose and lipids.

[0125] The results of ghrelin detection showed that, compared with the high-fat group, mice administered *Propionibacterium freundii* subsp. *S. sherry* PF310 bacterial suspension via gavage had a significantly lower serum ghrelin level by 41.26%. Intervention with inactivated *Propionibacterium freundii* subsp. *S. sherry* PF310 bacterial suspension also reduced ghrelin levels by 23.79%. Ghrelin is an orexin secreted by the stomach; its level increases during fasting and promotes food intake. PF310 significantly downregulated ghrelin levels, indicating that it can effectively inhibit orexin secretion and reduce the urge to eat. There were no significant differences between the control strain PF37 and the positive control group and the high-fat group.

[0126] A comprehensive analysis of the results from the four hormone tests revealed that PF310 exhibits a multi-target synergistic regulatory effect on energy metabolism-related hormones. By regulating GLP-1, leptin, ghrelin, and insulin, PF310 achieves a multi-faceted appetite-energy metabolism regulatory network, including suppressing appetite, delaying gastric emptying, enhancing satiety, promoting energy expenditure, and improving insulin resistance. This results in a comprehensive effect of reducing food intake, inhibiting weight gain, and improving lipid metabolism disorders, thereby preventing obesity caused by a high-fat diet. This discovery provides a novel probiotic resource and intervention strategy for the prevention and treatment of obesity.

[0127] Example 8: Study on the growth characteristics of Propionibacterium fischeri subsp. Schaeferi PF310 at 37℃

[0128] For probiotics to exert their physiological functions, they must first survive and maintain metabolic activity in the host's gut. The core temperature of the human body is approximately 37°C, while the standard culture temperature for *Propionibacterium freundii* subsp. *Schärni* is 30°C. This study aims to investigate the growth characteristics of *Propionibacterium freundii* subsp. *Schärni* PF310 at 37°C and evaluate its adaptability as an oral probiotic in the human body's temperature environment.

[0129] Following the method disclosed in our previous patent CN120173826B, the growth characteristics of *Propionibacterium freundii* subsp. *shee* PF310 at 37°C were investigated. *Propionibacterium freundii* subsp. *shee* PF310 strain was revived using fresh YEL medium. Four commercially available *Propionibacterium freundii* subsp. *shee* strains (CICC10284, ATCC9614, BNCC353332, PF-G68) and six other *Propionibacterium freundii* subsp. *shee* strains screened from cheese using the method described in the examples (PF37, PF051, PF087, PF135, PF172, and PF184) were selected as control strains. After one subculture, the seed culture was cultured to OD. 600 The inoculum concentration was 3.0. The seed culture was inoculated at a rate of 5% into a glass fermenter containing 2 L of YEL medium. The temperature was controlled at 37°C, pH 6.5, and stirring at 50 rpm for anaerobic fermentation for 96 hours. The OD of the culture medium was measured every 24 hours. 600 Absorbance value.

[0130] from Figure 6 It can be seen that, under the culture condition of 37℃, only *Propionibacterium fischeri* subsp. *Schärni* PF310 and PF37 showed good growth performance. At 72 hours of fermentation, the OD values ​​of PF310 and PF37... 600 The values ​​all reached around 30, significantly higher than other reference strains.

[0131] The above results indicate that Propionibacterium freundii subsp. Schaeferi PF310 and PF37 have similar optimal growth temperatures, both adapting to an environment of 37℃ (close to mammalian body temperature). This confirms that PF310 is a Propionibacterium strain that can adapt to human body temperature; increased temperature after colonization does not inhibit its growth, and the strain can still maintain good metabolic activity.

[0132] Based on the results of Examples 5-7, although PF310 and PF37 both exhibit similar temperature adaptability to 37°C, only PF310 possesses the function of regulating energy metabolism-related hormones and alleviating obesity. This further demonstrates that the unique function of PF310 cannot be attributed to temperature adaptability, but rather to the unique technical effects of this strain.

[0133] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0134] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Propionibacterium freudenreichii subsp. shermanii PF310, characterized in that, The strain has the preservation number CGMCC No. 31417.

2. A composition, characterized in that, It includes Propionibacterium fischeri subsp. Scheres PF310 as described in claim 1, and a pharmaceutically, food-, or feed-acceptable carrier.

3. The composition according to claim 2, characterized in that, The *Propionibacterium fischeri* subsp. Schaeferi PF310 exists in either live or inactivated form.

4. The use of Propionibacterium fischeri subsp. Scheres PF310 as described in claim 1, or the composition of any one of claims 2-3, in the preparation of products for the prevention and / or relief of obesity.

5. The application according to claim 4, characterized in that, The Propionibacterium fischeri subsp. Scheres PF310 or the composition of any one of claims 2-3 is used to regulate energy metabolism-related hormones, the regulation including one or more of the following: upregulation of glucagon-like peptide-1, upregulation of leptin, downregulation of ghrelin, and downregulation of insulin.

6. The use of Propionibacterium fischeri subsp. Scheres PF310 as described in claim 1, or the composition of any one of claims 2-3, in the preparation of a product for lowering triglycerides and / or total cholesterol.

7. The use of Propionibacterium fischeri subsp. Scheres PF310 as described in claim 1, or the composition of any one of claims 2-3, in the preparation of a product for improving insulin resistance.

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