Use of waddseaella for increasing intramuscular fat in animals

Abstract

The application discloses application of a ward's bacteria in improving intramuscular fat of animals and belongs to the field of microbiology. The application is based on 16S rRNA sequencing and macro-genome data integration analysis of excrement microorganisms of Laiwu pigs, Duroc pigs and Yorkshire pigs. Significant enrichment bacterial flora of each pig breed is screened through LEfSe differential analysis, and combined with joint analysis of two types of data, dominant flora with high abundance in Laiwu pigs is obtained, and the dominant flora includes the ward's bacteria. KEGG pathway annotation and enrichment analysis show that the ward's bacteria are significantly enriched in lipid metabolism related pathways such as fatty acid biosynthesis, unsaturated fatty acid biosynthesis and PPAR signal pathway. Animal experiment results show that supplementing the strain can improve the content of muscle tissue triglyceride, promote intramuscular fat deposition and up-regulate the expression of lipid synthesis related genes. The application can be used for preparing micro-ecological preparation or feed additive for improving intramuscular fat content of livestock and poultry and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to the application of Waldsartella in increasing intramuscular fat in animals. Background Technology

[0002] Intramuscular fat (IMF) refers to the fatty tissue deposited between muscle fibers and muscle bundles, and its content is one of the key indicators for evaluating meat quality. IMF levels directly affect the tenderness, juiciness, flavor, and consumer acceptance of meat. In modern livestock production, appropriately increasing the intramuscular fat content of animals has become an important goal for improving meat quality and enhancing the market value of meat products.

[0003] Currently, the main technical approaches to increasing intramuscular fat content in animals include genetic breeding and nutritional regulation. Genetic breeding involves selecting strains with high intramuscular fat traits, but this method is time-consuming, costly, and easily limited by the animal's genetic background. Nutritional regulation promotes lipid deposition by adjusting dietary energy levels, adding oils, or altering the nutrient structure; however, these methods often suffer from unstable effects, increased costs, and potential adverse effects on animal health in actual production. Therefore, developing safe, efficient, and stable new regulatory strategies has significant industrial value.

[0004] In recent years, the relationship between gut microbiota and host lipid metabolism has received widespread attention. Existing studies have shown that specific gut microbes can participate in the regulation of host lipid metabolism by producing short-chain fatty acids, regulating bile acid metabolism, and influencing signaling pathways related to fat synthesis and breakdown. Differences in intramuscular fat deposition capacity among different animal breeds are not only related to genetic factors but may also be closely related to differences in their gut microbiota structure. For example, there are significant differences in the gut microbiota composition between high-intracranial-fat pig breeds (such as Laiwu pigs) and low-intracranial-fat pig breeds (such as Duroc and Yorkshire pigs). However, current research on screening beneficial strains from gut microbes that clearly promote intramuscular fat deposition and verifying their effects remains very limited.

[0005] Sutterella wadsworthensis is an anaerobic bacterium widely found in the intestines of humans and animals, first isolated and described by Wexler et al. in 1996. Existing research on Sutterella wadsworthensis mainly focuses on its association with intestinal diseases (such as inflammatory bowel disease) and its bile acid resistance characteristics. To date, no functional reports have been found regarding Sutterella wadsworthensis's association with intramuscular fat deposition, lipid metabolism regulation, or meat quality improvement in animals. Whether this strain promotes intramuscular fat deposition, and its application value in livestock production, remain unexplored areas. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of *Ward's Sadella* in increasing intramuscular fat in animals. Based on a systematic analysis of the intestinal flora of different pig breeds, this invention is the first to discover and verify that *Ward's Sadella* (ATCC51579) has a novel function of increasing intramuscular fat in animals, providing a new microbial resource and application scheme for improving meat quality.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention leverages the phenotypic characteristics of the Laiwu pig, a local breed, which exhibits significantly high intramuscular fat content and excellent meat flavor. It integrates and analyzes 16S rRNA sequencing data and metagenomic data from fecal microorganisms of Laiwu, Duroc, and Yorkshire pigs from our laboratory and databases to compare differences in gut microbiota composition among different breeds. After standardized bioinformatics processing, the LEfSe (Linear Discriminant Analysis EffectSize) differential analysis method was used to screen for significantly different microbiota among different breeds, identifying highly abundant microbiota significantly enriched in the Laiwu pig gut. Furthermore, the 16S rRNA sequencing data and metagenomic sequencing data were jointly analyzed to screen for common microbiota that were predominantly enriched in Laiwu pigs in both datasets, thus improving the reliability of the screening results. This included *Waldesarterella*. Based on this, KEGG pathway annotation and enrichment analysis were performed on these dominant microbiota. The results showed that *Waldesarterella* was significantly enriched in lipid metabolism-related pathways, suggesting that it may participate in intramuscular fat deposition by regulating host lipid metabolism. Further animal experiments verified that supplementing with this strain can increase the triglyceride content in the muscle tissue of experimental animals and promote intramuscular lipid deposition, thereby increasing intramuscular fat levels.

[0008] In a first aspect, the present invention provides the application of *Ward's sartella* in increasing intramuscular fat in animals, wherein the application is as follows: (1) Direct application of Waldsartella salina to animals; or (2) Prepare a formulation of Waldessart bacteria for increasing intramuscular fat in animals.

[0009] The preservation number of the *Ward's sartella* strain is ATCC 51579.

[0010] The preparation is a microecological preparation or a feed additive.

[0011] The increase in intramuscular fat in animals includes at least one of the following: (a) Increases triglyceride content in muscle tissue; (b) Increase the proportion of lipid droplet area in muscle tissue; (c) Upregulates the expression of genes related to fatty acid synthesis in muscle tissue; (d) Downregulate the expression of genes related to lipolysis in muscle tissue.

[0012] In a second aspect, the present invention provides a microecological preparation for increasing intramuscular fat in animals, wherein the microecological preparation uses Waldsartella asiatica with accession number ATCC 51579 as the active ingredient.

[0013] Preferably, the microecological preparation also includes feed-grade acceptable carriers or excipients.

[0014] In a third aspect, the present invention provides a method for increasing intramuscular fat in an animal, comprising administering to the animal an effective amount of Waldsart bacteria with accession number ATCC 51579.

[0015] The effective dose is 1×10⁻⁶ per day. 6 ~1×10 10 CFU / kg animal body weight.

[0016] This invention uses a mouse animal model for gavage experiments to verify the effect of *Ward's Sadella* in increasing intramuscular fat in animals. Experimental results show that supplementation with *Ward's Sadella* can significantly increase the triglyceride content in animal muscle tissue, increase intramuscular fat, and has the potential to improve meat quality.

[0017] The beneficial effects of this invention are: This invention is the first to propose and verify that *Ward'sard* (ATCC 51579) can serve as a functional strain for increasing intramuscular fat in animals, expanding its application in the livestock industry. Research results show that supplementing with *Ward'sard* significantly promotes lipid deposition in animal muscle tissue and increases intramuscular fat levels, thereby improving meat quality. The strain described in this invention is derived from animal gut microbiota, exhibits high safety, and can be used as a microecological preparation or feed additive in animal production, demonstrating promising application prospects and industrialization value. Attached Figure Description

[0018] Figure 1 The results of differentially expressed microbiota analysis were obtained by integrating 16S rRNA sequencing and metagenomic data from fecal samples of Laiwu pigs (LW), Duroc pigs (DC), and Yorkshire pigs (YK) from our laboratory with data from publicly available databases. Figure A shows the genus-level differentially expressed microbiota obtained by LEfSe analysis based on 16S rRNA data; Figure B shows the genus-level differentially expressed microbiota obtained by LEfSe analysis based on metagenomic data.

[0019] Figure 2This is a Venn diagram constructed based on 16S rRNA sequencing and metagenomic sequencing results. Blue represents microorganisms with higher abundance in Laiwu pigs compared to Duroc and Yorkshire pigs in the metagenomic data; pink represents microorganisms with higher abundance in Laiwu pigs compared to Duroc and Yorkshire pigs in the 16S rRNA sequencing data. The overlapping area represents microorganisms with higher abundance in both datasets. The five microorganisms in the overlapping area are: g_Sutterella, g_Bacteroides, g_Sphingomonas, g_Acinetobacter, and g_Chlamydia.

[0020] Figure 3 The figure shows the results of functional prediction and pathway enrichment analysis using PICRUSt2 software in conjunction with the KEGG database.

[0021] Figure 4 The effect of gavage administration of *Ward's sartarius* on the body weight of mice was investigated. Figure A shows the trend of body weight change in mice of different treatment groups during gavage; Figure B shows the comparison of body weight gain in mice of different treatment groups during gavage. C represents the normal diet control group, S represents the *Ward's sartarius* bacterial solution gavage group, and F represents the high-fat diet group.

[0022] Figure 5 The results show the triglyceride (TG) content in mouse muscle, fat, and liver tissues. Figure a represents the TG content in muscle tissue; Figure b represents the TG content in fat tissue; and Figure c represents the TG content in liver tissue. The control group was a normal diet control group, the bacterial culture group was a group receiving *Ward's Sadella* bacterial culture via gavage, and the high-fat group was a group fed a high-fat diet.

[0023] Figure 6 Figure 16S rRNA sequencing results of fecal samples from mice after 28 days of gavage administration of *Sutterella waldsartine*. Figure a is a bar chart of LDA effect values ​​of differentially expressed bacterial genera in the three groups using LEfSe analysis; Figure b is a box plot comparing the relative abundance of *Sutterella* in the three groups; Figure c is a heatmap of differential expression clustering in multiple biological pathways between groups C and S. C represents the normal diet control group, S represents the *Sutterella waldsartine* gavage group, and F represents the high-fat feeding group.

[0024] Figure 7 The images show the results of Oil Red O staining of mouse TA muscle tissue. Image A shows the Oil Red O staining results of mouse TA muscle tissue in the normal diet control group; Image B shows the Oil Red O staining results of mouse TA muscle tissue in the group that was administered Waldersee bacteria via gavage.

[0025] Figure 8This image shows the results of qPCR detection of the expression levels of genes related to lipid metabolism in mouse muscle tissue. The control group represents the normal diet control group; the gavage group represents the group that received *Ward's salt* bacterial solution via gavage; and the high-fat group represents the group that was fed a high-fat diet. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] The Waldsart bacteria used in the examples have the accession number ATCC 51579 and were purchased from the American Type Culture Collection (ATCC).

[0028] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0029] Example 1: Screening of Differential Microbiota and Identification of Target Bacteria We selected 16S rRNA sequencing data and metagenomic data from fecal samples of Laiwu pigs (high intramuscular fat breed), Duroc pigs (low intramuscular fat breed), and Yorkshire pigs (low intramuscular fat breed) from our laboratory and from our database as the research subjects. The above data were downloaded and organized, and data quality was screened and standardized.

[0030] The LEfSe (linear discriminant analysis effect size) method was used to compare the differences in species composition of the gut microbiota among the three groups of pigs, with an LDA threshold of ≥2.0. Venn diagrams were further constructed to screen for microbial taxa that were significantly enriched only in the gut of Laiwu pigs, but extremely low in abundance or absent in Duroc and Yorkshire pigs. The results showed that *Sutterella wadsworthensis* was specifically enriched in the gut of Laiwu pigs. Figure 1 , Figure 2 ).

[0031] Metabolic function was predicted using PICRUSt2 software with 16S rRNA sequencing data as input and the KEGG database. The abundance of KEGG pathways in *Ward'sard* was compared with that of other enriched strains. Results showed that *Ward'sard* was mainly enriched in pathways such as unsaturated fatty acid biosynthesis, fatty acid biosynthesis, and fatty acid metabolism compared to other microorganisms. Based on this, this strain was identified as a candidate functional strain.

[0032] Example 2: Animal Intervention Experiment The Sutterella wadsworthensis ATCC 51579 used in this invention is derived from a commercial bacterial suspension and is provided by a commercial supplier.

[0033] A commercially available bacterial suspension of *Sutterella wadsworthensis* ATCC 51579 was used as the bacterial source for the experiment, with a nominal bacterial concentration of 1 × 10⁻⁶. 8 CFU / mL. Before use, thoroughly mix the bacterial suspension and administer directly to animals via gavage. The bacterial suspension can be stored at 4°C for a short period (not exceeding 1 week); bring it to room temperature and mix well before use.

[0034] Thirty 6-week-old female C57BL / 6 mice, weighing 17–19 g, were randomly divided into three groups of 10 mice each: group C, group S, and group F. Among them: Group C (normal control group): fed with normal feed and administered 200ul sterile PBS by gavage daily; Group S (Ward's Sadella group): fed with normal feed, and administered 200 μL daily via gavage at a concentration of 1×10⁻⁶. 8 CFU / ml Waldsartella suspension; Group F (high-fat feeding group): fed with high-fat diet and administered 200ul sterile PBS by gavage daily.

[0035] Table 1: Formulation of ordinary and high-fat feeds used in the experiment Mice in each group were administered gavage at fixed times, from 6 weeks of age to 9 weeks of age, once every other day for 4 consecutive weeks. During the experiment, the mice were housed in a diurnal environment (light hours from 9:00 to 21:00), and their activity level, behavior, coat luster, food intake, and general health were observed and recorded daily. Throughout the experiment, no abnormalities such as disease, death, vomiting, or diarrhea were observed in any group of mice, and no significant abnormalities were found in their behavior, mental state, coat condition, or food intake.

[0036] Each mouse was weighed at a fixed time each week (Monday morning), and weight changes were recorded. At the end of the experiment (day 28), the mice were weighed again, and weight gain (final weight - initial weight) was calculated. Results are as follows: Figure 4 As shown, compared with group C, the weight gain of mice in groups S and F both showed an increasing trend. This result indicates that gavage with *Ward's sartans* had no significant adverse effects on mouse growth and may have a certain regulatory effect on the body's energy metabolism.

[0037] Example 3: Fecal sample collection and verification of gut microbiota colonization Based on Example 2, fresh feces from mice in each group were collected on day 0 (before intervention), day 14 (mid-intervention), and day 28 (end of intervention) after gavage intervention. 300 mg of feces were collected from each mouse. The samples were immediately numbered and labeled after collection, flash-frozen in liquid nitrogen, and then stored at -80°C for later use.

[0038] Fecal DNA was extracted using the same method as in Example 1, and 16S rRNA gene sequencing analysis was performed on the above fecal samples to evaluate changes in the gut microbiota structure. Figure 6 As shown, compared with the control group and the high-fat feeding group, the relative abundance of *Ward'sard* in mice administered *Ward'sard* via gavage showed an increasing trend, indicating that the exogenously administered *Ward'sard* successfully colonized the intestine. Further functional prediction and pathway enrichment analysis of the sequencing data revealed that the gavage group showed an enrichment trend in lipid-related metabolic pathways, such as the peroxisome proliferator-activated receptor (PPAR) signaling pathway and the adipokine signaling pathway. These results suggest that *Ward'sard* intervention may be related to the regulation of lipid metabolism-related signaling pathways.

[0039] Example 4: Detection of tissue triglyceride content After the gavage experiment in Example 2, mice in each group were euthanized by cervical dislocation, and muscle, adipose, and liver tissues were collected and labeled. The collected tissue samples were flash-frozen in liquid nitrogen and then stored at -80°C for later use. The TG content in each tissue sample was detected and analyzed using a triglyceride (TG) assay kit (single-reagent GPO-PAP method). The test results are as follows: Figure 5 As shown, in muscle tissue, compared with the control group, the triglyceride content in mice administered *Ward'sard* via gavage and in the high-fat feeding group was significantly increased. In adipose tissue, the triglyceride content in mice administered *Ward'sard* via gavage and in the high-fat feeding group was slightly increased compared with the control group, but the difference did not reach a statistically significant level. In liver tissue, only the triglyceride content in the high-fat feeding group was significantly increased, while no significant change was observed in the *Ward'sard* via gavage group. These results indicate that *Ward'sard* via gavage can specifically promote the accumulation of triglycerides in muscle tissue without causing fat deposition in the liver.

[0040] Example 5: Histological examination of intramuscular fat deposition in muscle tissue After the gavage experiment in Example 2, mice in each group were euthanized according to animal experimental procedures, and the tibialis anterior muscle (TA muscle) tissue was isolated and collected. The muscle tissue was embedded in an embedding medium (OCT), then flash-frozen in liquid nitrogen, and frozen sections were prepared for later use. The frozen sections were stained with Oil Red O, a commonly used lipid-specific staining agent that binds to triglycerides and other neutral lipids in tissues, turning them red, thus enabling the detection and localization analysis of lipid deposition. The staining results are as follows: Figure 7 As shown, compared with the control group, the proportion of lipid droplet area in the TA muscle tissue of mice administered *Ward'sardella* via gavage was significantly increased. This histological result further confirms that *Ward'sardella* gavage can significantly promote intramuscular fat deposition.

[0041] Example 6: Detection of expression levels of lipid metabolism-related genes After the gavage experiment in Example 2, mice were euthanized by cervical dislocation. Muscle tissue from each group was rapidly isolated, labeled, and immediately flash-frozen in liquid nitrogen, then transferred to a -80°C freezer for later storage. Total RNA was extracted from the muscle tissue of each group using a commercially available total RNA extraction kit. After passing quality testing, cDNA was synthesized via reverse transcription. Using the obtained cDNA as a template, the expression levels of lipid metabolism-related genes were detected by real-time quantitative PCR (qPCR) using the SYBR Green fluorescent dye method. The results showed that the expression levels of fatty acid synthesis genes (FASN, ACSL3, ACLY, ACC-α) in the gavage group were significantly higher than those in the control group; among unsaturated fatty acid synthesis genes (ELOVL6, SCD1, FADS1), the expression level of FADS1 was significantly increased in the gavage group; the expression levels of neutral lipid-related genes (DGAT2, PPARγ, PLIN2) were significantly increased in the gavage group; the expression level of the lipid oxidation-decomposition gene (ATGL) was significantly decreased in the gavage group, while the expression levels of the remaining decomposition-related genes (HSL, LPL, UCP-2) were not significantly different from those in the control group. These gene expression profiles revealed the mechanism by which *Ward'sard* promotes intramuscular fat deposition at the molecular level: mainly through activating the de novo fatty acid synthesis pathway and inhibiting the lipid decomposition pathway.

[0042] Table 2: Primer sequences for real-time quantitative PCR of lipid metabolism-related genes The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. The application of *Ward's sartella* in increasing intramuscular fat in animals, characterized in that... The application is as follows: (1) Direct application of Waldsartella salina to animals; or (2) Prepare a formulation of Waldsart bacteria for increasing intramuscular fat in animals; The increase in intramuscular fat in animals includes at least one of the following: (a) Increases triglyceride content in muscle tissue; (b) Increase the proportion of lipid droplet area in muscle tissue; The preservation number of the *Ward's sartella* strain is ATCC 51579.

2. The application according to claim 1, characterized in that, The preparation is a microecological preparation or a feed additive.

3. The application according to claim 1, characterized in that, A formulation for increasing intramuscular fat in animals was prepared using Waldsartella vulgaris (accession number ATCC 51579) as the active ingredient.

4. The application according to claim 3, characterized in that, The formulation also includes feed-grade acceptable carriers or excipients.

5. The application according to claim 1, characterized in that, Intramuscular fat in animals was increased by administering an effective amount of Waldsart bacteria with accession number ATCC51579.

6. The application according to claim 5, characterized in that, The effective dose of *Ward's serrata* with accession number ATCC 51579 is 1×10⁻⁶ per day. 6 ~1×10 10 CFU / kg animal body weight.

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