Application of a strain of *Bruchwegia bournei* in the preparation of products that promote intramuscular fat deposition in livestock and poultry

Abstract

This invention provides the application of a *Breago brevis* strain in the preparation of products that promote intramuscular fat deposition in livestock and poultry. The *Breago brevis* strain is *Breago brevis* JCM 32280. Under high-fiber dietary conditions, supplementation with *Breago brevis* JCM 32280 of this invention can efficiently degrade fiber to produce high concentrations of butyrate. Butyrate, as a key effector molecule, can directly bind to and activate PPARγ receptors in host cells, thereby upregulating the MOGAT2-mediated triglyceride synthesis pathway, significantly increasing body fat percentage in mammals and promoting intramuscular fat (marbling). Simultaneously, this strain can reshape the intestinal microecology, specifically increasing the abundance of beneficial intestinal lactobacilli and enhancing the intestinal mucus barrier, providing a safe and efficient microbial preparation strategy for improving the fat deposition capacity and meat flavor of livestock and poultry.

Description

Technical Field

[0001] This invention relates to the fields of microbial applications and biotechnology, specifically to the application of a strain of *Bruchwegia bournei* in the preparation of products that promote intramuscular fat deposition in livestock and poultry. Background Technology

[0002] Intramuscular fat (IMF) content is a key indicator determining the quality of meats such as pork (e.g., tenderness, flavor, and juiciness). In livestock production, appropriately increasing intramuscular fat deposition (commonly known as "marbling") is an important means of enhancing the economic value of meat products.

[0003] Gut microbiota are considered a "second genome" regulating host metabolism. *Breospira brevicornu* (… Treponema bryantii This strain is a spirochete found in the pig intestine and has the ability to degrade fiber. However, current technology has not revealed the causal effect of this strain on intramuscular fat deposition under specific dietary conditions, nor has it reported its regulatory role and molecular interaction mechanism on intestinal symbiotic flora (such as lactobacilli). Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide an application of a *Bretorius* strain in the preparation of products that promote intramuscular fat deposition in livestock and poultry. Under high-fiber dietary conditions, supplementation with *Bretorius* strain JCM 32280 of this invention can efficiently degrade fiber to produce high concentrations of butyric acid. Butyric acid, as a key effector molecule, can directly bind to and activate PPARγ receptors in host cells, thereby upregulating the MOGAT2-mediated triglyceride synthesis pathway, significantly increasing body fat percentage in mammals and promoting intramuscular fat (marbling) deposition. Simultaneously, this strain can reshape the intestinal microecology, specifically increasing the abundance of beneficial bacteria *Lactobacillus intestinalis* and enhancing the intestinal mucus barrier (MUC2), providing a safe and efficient microbial preparation strategy for improving the fat deposition capacity of livestock and poultry and enhancing meat flavor.

[0005] Technical solution: The application of a strain of *Bryonymus bournei* in the preparation of products that promote intramuscular fat deposition in livestock and poultry, wherein the strain of *Bryonymus bournei* is *Bryonymus bournei* JCM 32280.

[0006] Furthermore, the application needs to be carried out under conditions of a high-fiber diet.

[0007] Furthermore, the promotion of intramuscular fat deposition in livestock and poultry specifically includes one or more of the following: (1) Increase the body fat percentage of mammals; (2) Increase intramuscular fat deposition or marbling score in mammals; (3) Promotes fat synthesis and accumulation in mammalian white adipose tissue; (4) Increases the level of butyrate in the blood circulation of mammals; (5) Activate the triglyceride synthesis pathway mediated by monoacylglycerol O-acyltransferase 2 in adipose tissue; (6) It regulates the structure of the intestinal flora and specifically increases the relative abundance of intestinal lactobacilli; (7) Enhances intestinal mucus barrier function and upregulates the expression of mucin MUC2.

[0008] Furthermore, the products include feed additives, microbial ecological preparations, or veterinary compositions.

[0009] A method for promoting intramuscular fat deposition in livestock and poultry involves feeding the livestock and poultry a high-fiber feed and simultaneously administering an effective dose of Leptospira Brinelliae JCM 32280.

[0010] Furthermore, the effective dose is 10. 9 CFU / mL. Beneficial effects

[0011] Significant fat deposition effect: Under high-fiber diet (HFiD) conditions, this strain can significantly promote the expansion of adipose tissue, greatly increase the body fat percentage, and is independent of significant weight gain; 2. Specific intramuscular fat enhancement: Histological examination confirmed that treatment with this strain could induce significant deposition of lipid droplets between muscle fibers; 3. Optimized gut microbiota structure: 16S rRNA sequencing confirmed that intervention with this strain can significantly improve the uniformity of the gut microbiota and specifically enrich Lactobacillus intestinalis, thereby improving the gut microenvironment; 4. Clear molecular mechanism: (1) Metabolic drive: The strain efficiently degrades dietary fiber and specifically increases serum butyrate levels; (2) Target activation: Butyric acid, as a ligand, directly binds to the ligand-binding domain (LBD) of PPARγ, inducing a conformational change and activation. (3) Pathway regulation: Activated PPARγ significantly upregulates the expression of MOGAT2, accelerating the synthesis and storage of triglycerides. Attached Figure Description

[0012] Figure 1 Figure showing the changes in body fat percentage in mice fed a high-fiber diet (HFiD) and those treated with bacterial strain intervention (HFiD+TB). Figure 2Photographs and graphs showing changes in adipose tissue weight of epididymal fat (eWAT) and inguinal fat (iWAT) in mice fed a high-fiber diet (HFiD) and with bacterial strain intervention (HFiD+TB); Figure A shows epididymal fat (eWAT); Figure B shows inguinal fat (iWAT). Figure 3 Images of epididymal fat (eWAT) and inguinal fat (iWAT) tissue sections (H&E staining) from mice fed a high-fiber diet (HFiD) and strain intervention group (HFiD+TB); Figure 4 Images showing intramuscular fat (IMF) deposition in mice fed a high-fiber diet (HFiD) and those treated with bacterial strains (HFiD+TB) (H&E staining). Figure 5 Images showing intramuscular fat (IMF) deposition in mice fed a high-fiber diet (HFiD) and those treated with bacterial strains (HFiD+TB) (Oil Red O staining). Figure 6 Image showing intramuscular fat (IMF) deposition in mice fed a high-fiber diet (HFiD) and those treated with bacterial strains (HFiD+TB) (immunofluorescence staining). Figure 7 The concentration of butyrate in the serum of mice in the high-fiber diet (HFiD) and strain intervention group (HFiD+TB) was measured. Figure 8 The concentrations of ten other short-chain fatty acids in the serum of mice fed a high-fiber diet (HFiD) and those treated with bacterial strain intervention (HFiD+TB): propionic acid, acetic acid, isobutyric acid, isovaleric acid, valeric acid, caprylic acid, nonanoic acid, hexanoic acid, heptanoic acid, and decanoic acid. Figure 9 Simpson index for mice in the high-fiber diet (HFiD) and strain intervention group (HFiD+TB); Figure 10 PCoA analysis of mice fed a high-fiber diet (HFiD) and those in the bacterial strain intervention group (HFiD+TB); Figure 11 For species difference analysis (LEfSe); Figure 12 The graph shows the changes in intracellular triglyceride (TG) content in the butyric acid-treated group. Figure 13 Images of lipid droplets stained with Bodipy fluorescence, images of cell nuclei stained with Hoechst, and images of lipid droplets stained with ORO in butyrate-treated cells. Figure 14The results showed changes in MOGAT2 and PPARγ by qPCR (mRNA expression) and Western Blot (protein expression) in the butyric acid-treated group, as well as changes in the protein expression levels of key molecules in the PPARγ signaling pathway, ADIPOQ, FABP4, and PLIN1. Figure 15 This is a schematic diagram of the molecular docking mode between butyric acid and PPARγ LBD. Figure 16 Images of colon tissue sections from mice fed a high-fiber diet (HFiD) and those in the bacterial strain intervention group (HFiD+TB); Figure 17 qPCR detection for mice on high-fiber diet (HFiD) and strain intervention group (HFiD+TB). Detailed Implementation

[0013] This invention proposes the application of a strain of *Treponema bournei* in the preparation of products that promote intramuscular fat deposition in livestock and poultry. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific examples. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0014] All raw materials and reagents used in the embodiments of this invention are commercially available. Statistical analysis methods used in this invention: data are expressed as Mean ± SEM. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P<0.05 It is considered to be statistically significant.

[0015] The strain used in this embodiment is *Treponema bournei* (…). Treponema bryantii The strain, numbered JCM32280, was purchased from the Japan Collection of Microorganisms (JCM).

[0016] Example 1: Cultivation and preparation of Treponema bryantii strain Step 1: Culture medium formulation (modified rumen fluid medium) To meet the strict anaerobic growth requirements of this strain, the following culture medium is prepared: (1) Basic components: glucose, yeast extract, protein powder, sodium acetate, mineral salt solution; (2) Reducing agent: L-cysteine ​​is added as a reducing agent to maintain a low redox potential; (3) pH adjustment: Adjust the pH to 6.7-7.0 using Na2CO3; (4) Preparation method: Prepared under strict anaerobic conditions (CO2 gas is introduced for 40 minutes to remove oxygen), and sterilized at 121°C for 15 minutes; Step 2: Preparation of bacterial suspension: The frozen bacterial suspension... Treponema bryantii JCM 32280 was inoculated into the above liquid medium and cultured in an anaerobic workstation at 39°C until the logarithmic growth phase. The bacterial cells were collected, centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The cells were washed twice with sterile anaerobic phosphate-buffered saline (PBS, pH 7.4), and finally resuspended in sterile PBS to adjust the bacterial concentration to 10. 9 The bacterial agent was prepared at CFU / mL for animal experiments.

[0017] Example 2: Establishment of a high-fiber diet intervention model 1. Experimental animals: Seven-week-old male C57BL / 6J mice (purchased from Vital Rivers, Beijing) were selected and acclimatized for one week under specific pathogen-free (SPF) environment, constant temperature (25 ± 2℃), constant humidity (50% ± 5%), and 12-hour light / dark cycle conditions. 2. This embodiment provides an experimental animal feed that simulates the dietary structure of roughage-tolerant animals (such as ruminants or herbivorous livestock). Specifically, the dietary fiber source is cellulose, and the amount added is 8% of the total weight, so as to significantly increase the crude fiber content in the feed while maintaining the relative balance of basic nutrients.

[0018] Feed formulation composition: The specific raw material composition and weight percentage are shown in the table below: Table 1: Comparison of High-Fiber Diet (HFiD) and Normal Maintenance Diet (NCD) Formulations (by weight %)

[0019] Preparation process: Weigh all raw materials according to the above formula. First, crush the bulk raw materials such as corn, wheat, and soybean meal and pass them through a 40-mesh sieve. Then, gradually expand the mixing of microcrystalline cellulose with premix (vitamins, minerals, and amino acids) to ensure that the cellulose is evenly distributed in the feed. Finally, mix all components, add an appropriate amount of water for conditioning, granulate at 70-80°C, dry until the moisture content is less than 10%, and vacuum package after ultraviolet sterilization for later use.

[0020] Nutritional analysis: After adjusting the above formula, the main nutritional parameters of this high-fiber feed are as follows: Crude Protein: ≥ 18% Crude Fat: ≥ 4% Crude fiber: Approximately 12-14% (significantly higher than the 4-5% of ordinary feed) Total Dietary Fiber: ≥ 20% 3. Experimental grouping and treatment: After the adaptation period, 24 mice were randomly divided into two groups (n=12 in each group). Both groups of mice were fed the above-mentioned high-fiber diet (HFiD) throughout the entire experiment. The specific treatments are as follows: (1) High-fiber diet control group (HFiD-Control): 0.2 mL of sterile phosphate-buffered saline (PBS) was administered by gavage every 3 days as a blank control; (2) High-fiber diet treatment group (HFiD-TB): 0.2 mL of Treponema bryantii (JCM 32280) bacterial suspension was administered by gavage every 3 days, with a bacterial concentration of 10. 9 CFU / mL; 4. Experimental period: The experiment was conducted for 8 weeks. During this period, the mice had free access to food and water. Their weight and food intake were recorded weekly to monitor their growth and energy intake. Results analysis:

[0021] 1. The effect of promoting the deposition of body fat and intramuscular fat under high-fiber diet conditions. 1.1 Effect on body fat percentage: Analysis using a mouse body composition analyzer (MesoQMR) revealed (e.g.) Figure 1 As shown in the figure, under high-fiber diet (HFiD) conditions, the body fat percentage (Body Fat %) of mice in the HFiD-TB group was significantly increased compared to the control group (P = 0.011). This confirms that the fat deposition-promoting effect of this strain is "fiber-dependent".

[0022] 1.2 Adipose tissue weight: Dissection and weighing showed that the weight of white adipose tissue was significantly increased in the HFiD+TB group mice: (1) Epididymal fat (eWAT): The weight was significantly higher than that of the control group (P = 0.009) (e.g. Figure 2 (as shown) (2) Inguinal fat (iWAT): The weight was significantly higher than that of the control group (P = 0.014) (e.g. Figure 2 As shown), tissue sections (H&E staining) revealed that adipocytes in the TB group were enlarged (hypertrophic) and their lipid droplets were plump (as shown). Figure 3 (As shown).

[0023] 1.3 Detection of Intramuscular Fat (IMF) Deposition: Histological analysis was performed on mouse quadriceps femoris muscles. (1) H&E staining: Obvious adipocyte infiltration was observed between myofibrils in the HFiD+TB group (e.g. Figure 4 (as shown) (2) Oil Red O staining: shows a significant increase in the area of ​​red-stained neutral lipid droplets between muscle bundles (e.g., Figure 5 (as shown) (3) Immunofluorescence staining: Laminin (labeling sarcolemma) and PLIN1 (peripheral lipid droplet protein, labeling lipid droplets) were used for double staining. The results showed that the density of PLIN1-positive lipid droplets in the muscle tissue of the TB group was significantly higher than that in the control group (e.g., Figure 6 (As shown).

[0024] The above results confirm that this strain can effectively promote intramuscular fat deposition, mimicking the phenotype of improved meat texture (marbling).

[0025] 2. Serum targeted metabolomics analysis (identification of butyric acid) 2.1 Test methods: Mouse serum was collected, and 11 short-chain fatty acids (SCFAs) were quantitatively analyzed using UHPLC-MS / MS technology (including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, etc.).

[0026] 2.2 Quantitative results showed that, compared with the control group, the serum butyric acid concentration in the HFiD+TB group was significantly increased (P = 0.008). Figure 7 As shown in the figure, the metabolite with the most significant changes (highest Fold Change) was acetic acid, propionic acid, isobutyric acid, valeric acid, and other short-chain fatty acids showed no statistically significant differences in serum concentrations (P>0.05). Figure 8 (As shown). Butyric acid was confirmed to be a key hematopoietic effector molecule mediating the lipotropic effects of the strain.

[0027] 3. Gut microbiota remodeling based on 16S rRNA sequencing 3.1 Sequencing and Analysis: DNA was extracted from mouse feces, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina platform; 3.2 Diversity Analysis (1) Alpha diversity: The Simpson index was significantly lower in the HFiD+TB group (P = 0.028), indicating that the gut microbiome had been remodeled; the Shannon index showed no significant difference (P = 0.058), indicating that the intervention remodeled the community structure by changing the relative abundance of specific taxa, but did not reduce the overall species richness (e.g., Figure 9 (As shown).

[0028] (2) Beta diversity: Principal coordinate analysis (PCoA) showed a significant separation of the gut microbiota structure between the TB group and the control group (PERMANOVA, P<0.05), indicating that bacterial colonization significantly altered the gut microbiota structure (e.g., Figure 10 (As shown).

[0029] 3.3 Species difference analysis (LEfSe) and linear discriminant analysis (LDA Score > 3.0) were used to screen for differentially expressed bacterial groups. The results showed that the HFiD+TB group was significantly enriched with the following beneficial bacterial groups: (1) Family level: Significant enrichment in Lactobacillaceae; (2) Species level: The relative abundance of *Lactobacillus intestinalis* was significantly increased. This indicates that colonization of *Treponema bryantii* can reshape the intestinal environment and promote the symbiosis and proliferation of specific lactic acid bacteria (e.g., *Lactobacillus intestinalis*). Figure 11 (As shown).

[0030] 4. Molecular mechanism verification—butyric acid-PPARγ-MOGAT2 axis 4.1 Cell model validation (3T3-L1) (1) Treatment: Add 1 mM sodium butyrate to the 3T3-L1 preadipocyte differentiation induction solution. (2) Phenotype: The intracellular triglyceride (TG) content in the butyric acid-treated group was significantly increased (e.g. Figure 12 As shown), Bodipy staining and ORO staining revealed increased lipid droplet accumulation (e.g. Figure 13 (as shown) (3) Gene expression: qPCR and Western blot analysis confirmed that butyrate treatment significantly upregulated the mRNA and protein expression levels of PPARγ (peroxisome proliferator-activated receptor PPARγ) and MOGAT2 (monoacylglycerol O-acyltransferase 2), while also upregulating downstream effector proteins ADIPOQ, FABP4, and PLIN1 (e.g., Figure 14 (As shown).

[0031] 4.2 Target confirmation (molecular docking) (1) Molecular Docking: Structural simulations show that butyrate acts as a ligand, binding to the ligand-binding domain (LBD) of PPARγ and forming hydrogen bonds with key amino acid residues Tyr464, His440, and Tyr314, thus stabilizing the active conformation of PPARγ (e.g., Figure 15 (As shown).

[0032] 5. Enhancement of intestinal barrier function 5.1 Histological Examination: HE staining of mouse colon tissue showed that the HFiD+TB group did not cause damage to the colon, and there were no morphological or pathological differences between the two groups. AB-PAS (allicin blue-periodic acid Schiff) staining of mouse colon tissue sections showed that the HFiD+TB group had an increased number of goblet cells and increased mucin secretion (e.g., ...). Figure 16 (as shown) 5.2 Molecular Detection (1) Immunohistochemistry: showed that the protein expression of MUC2 (mucin 2) on the surface of the colonic epithelium in the TB group was significantly enhanced. Figure 16 ); (2) qPCR detection: TB intervention significantly upregulated the expression of mucus synthesis gene MUC2 and glycosylation modification gene GCNT3 (P<0.05), and simultaneously activated calcium ion signaling pathway-related genes (RYR3, CHRM3) (e.g. Figure 17 As shown in the figure, this strain promotes fat deposition while protecting gut health by enhancing the mucus barrier.

[0033] In summary, the embodiments of the present invention fully demonstrate that Brinell spirochete ( Treponema bryantii JCM 32280, when combined with a high-fiber diet, produces butyric acid through fermentation. After entering the bloodstream, butyric acid directly binds to and activates PPARγ in adipose tissue, thereby upregulating MOGAT2-mediated triglyceride synthesis, thus increasing body fat percentage and intramuscular fat deposition. At the same time, it optimizes the gut microbiota structure (enriches lactobacilli) and enhances the intestinal barrier function.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a strain of *Bruchwegia bournei* in the preparation of products that promote intramuscular fat deposition in livestock and poultry, characterized in that, The strain of *Bryonymus bournei* is *Bryonymus bournei* JCM 32280.

2. The application according to claim 1, characterized in that, The application needs to be performed under conditions of a high-fiber diet.

3. The application according to claim 1 or 2, characterized in that, The promotion of intramuscular fat deposition in livestock and poultry specifically includes one or more of the following: (1) Increase the body fat percentage of mammals; (2) Increase intramuscular fat deposition or marbling score in mammals; (3) Promotes fat synthesis and accumulation in mammalian white adipose tissue; (4) Increases the level of butyrate in the blood circulation of mammals; (5) Activate the triglyceride synthesis pathway mediated by monoacylglycerol O-acyltransferase 2 in adipose tissue; (6) It regulates the structure of the intestinal flora and specifically increases the relative abundance of intestinal lactobacilli; (7) Enhances intestinal mucus barrier function and upregulates the expression of mucin MUC2.

4. The application according to claim 1, characterized in that, The products include feed additives, microbial ecological preparations, or veterinary compositions.

5. A method for promoting intramuscular fat deposition in livestock and poultry, characterized in that, Livestock and poultry should be fed a high-fiber diet and simultaneously given an effective dose of Breech-Burney's spirochete JCM 32280.

6. The method according to claim 5, characterized in that, The effective dose is 10 9 CFU / mL.

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