Clostridium butyricum ML5358 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses clostridium butyricum ML5358 and application thereof.The clostridium butyricum ML5358 provided by the invention has the preservation number of CGMCC No.37000. The strain can effectively relieve mouse colitis induced by DSS and remarkably reduce expression of colon tissue inflammatory factors Il-6 and Il-1beta, and the preservation number of the clostridium butyricum ML5358 is CGMCC No.37000. The clostridium butyricum ML5358 is CGMCC No.37000. The clostridium butyricum ML5358 is CGMCC No.37000. In addition, skeletal muscle lipid metabolism can be regulated and controlled through the intestine-muscle axis, expression of fat synthesis and uptake related genes Srebf1, Acc1 and Fabp4 is specifically up-regulated, the content of intramuscular triglyceride is remarkably increased, and intramuscular fat deposition is promoted. According to the clostridium butyricum ML5358 and the application thereof, the functional limitation of traditional clostridium butyricum is broken through, and a new strain resource is provided for developing a novel microbial preparation or a feed additive capable of improving intestinal health and improving livestock and poultry meat quality.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to Clostridium butyricum ML5358 and its applications. Background Technology

[0002] Meat quality is a key indicator for measuring livestock and poultry production performance and economic benefits. Among them, intramuscular fat content is the core element that determines the flavor, tenderness, and juiciness of meat. Under modern intensive farming models, many economic animals (such as lean commercial pigs and fast-growing poultry) generally suffer from insufficient intramuscular fat deposition, resulting in dry meat and poor flavor.

[0003] Increasing intramuscular fat content through genetic selection and other methods has limitations such as long development cycles, high costs, and unstable effects. Studies have shown that gut health is closely related to systemic metabolism, and the "gut-muscle axis" theory suggests that gut inflammation and dysbiosis may affect skeletal muscle lipid metabolism through systemic signal transduction. Therefore, starting with gut microbiota intervention, seeking interventions that can simultaneously maintain the intestinal barrier and positively regulate intramuscular fat metabolism has become a potential new strategy for improving meat quality.

[0004] Clostridium butyricum is a strictly anaerobic, spore-forming Gram-positive bacterium that has attracted much attention due to its ability to produce butyric acid and other bioactive metabolites. Previous studies have shown that supplementation with Clostridium butyricum can enhance intestinal barrier function, thereby alleviating colitis. Furthermore, Clostridium butyricum can upregulate the expression of genes related to lipid synthesis, thereby increasing intramuscular fat deposition. However, whether Clostridium butyricum can directly and efficiently promote intramuscular fat deposition through the gut-muscle axis, while simultaneously alleviating intestinal inflammation, thus fundamentally improving the quality of livestock and poultry meat, remains unreported. Summary of the Invention

[0005] The purpose of this invention is to overcome the technical problem of the single function of Clostridium butyricum products in the prior art, and to provide a Clostridium butyricum strain ML5358 isolated from fecal samples of Chinese local pigs (Guangdong Small-eared Pig). This strain not only has good tolerance to gastric acid and bile salts, but also shows the unique potential to effectively alleviate colitis and regulate the expression of skeletal muscle lipid metabolism genes in mammalian models.

[0006] To achieve the above objectives, the present invention provides Clostridium butyricum ML5358, which has the accession number CGMCC No.37000 and the accession date of December 10, 2025.

[0007] A microbial agent is also provided, comprising Clostridium butyricum ML5358 or the fermentation product of Clostridium butyricum ML5358.

[0008] A feed additive is also provided, comprising Clostridium butyricum ML5358 or a bacterial agent.

[0009] Furthermore, based on the weight or volume of the feed additive, the viable count of Clostridium butyricum ML5358 is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0010] It also provides the application of feed additives in the preparation of products used to increase intramuscular fat content in animals.

[0011] It also provides the use of feed additives in the preparation of products for the prevention and / or relief of intestinal inflammation in animals.

[0012] Furthermore, the product can reduce the expression levels of inflammatory factors Il-6 and Il-1β in colon tissue.

[0013] It also provides the application of feed additives in the preparation of products for regulating lipid metabolism in animal skeletal muscle.

[0014] Furthermore, regulation of skeletal muscle lipid metabolism includes promoting the expression of genes related to fat synthesis and uptake.

[0015] Furthermore, genes related to fat synthesis and uptake include at least one of Srebf1, Acc1, and Fapp4.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The core advantage of Clostridium butyricum ML5358 provided by this invention lies in the fact that it has been verified for the first time in a mammalian model to maintain intestinal health and promote intramuscular lipid deposition through the "gut-muscle axis". This breaks through the functional limitations of traditional Clostridium butyricum, which is mainly used to prevent diarrhea and promote growth. It provides a brand-new strain resource and key scientific basis for the development of new feed additives for improving the health and meat quality of economic animals.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 The results of 16S rDNA comparison of Clostridium butyricum ML5358 in the embodiments of the present invention; Figure 2 The morphological identification results of Clostridium butyricum ML5358 in the embodiments of the present invention are shown in Figure A, which shows the colony morphology of Clostridium butyricum ML5358 after 24 hours of culture on RCM solid medium, and B shows the bacterial morphology observed under an oil immersion microscope (scale bar 20 micrometers) after Gram staining. Figure 3The figures show the growth characteristics of Clostridium butyricum ML5358 as described in this invention. Figure A represents the growth curve (OD) of the strain. 600 B is the curve showing the change in viable cell count, and C is the curve showing the change in pH value during the culture process; Figure 4 The figures show the in vitro tolerance test results of Clostridium butyricum ML5358 in this embodiment of the invention. Figure A represents the acid tolerance survival rate of the strain under different pH conditions (pH 2, 3, and 4), and Figure B represents the bile salt tolerance survival rate of the strain in different concentrations (0.1%, 0.2%, and 0.3%) of bile salts. Different lowercase letters (a, b, c) indicate significant differences between groups. P< 0.05 The same letter indicates that there is no significant difference between groups. P >0.05); Figure 5 This refers to the relative expression levels of inflammatory factors TNF-α, Il-1β, and Il-6 in the colonic tissue of mice with DSS-induced colitis by Clostridium butyricum ML5358 in this embodiment of the invention; where different lowercase letters (a, b) indicate significant differences between groups. P <0.05, with the same letter indicating no significant difference between groups ( P >0.05); Figure 6 The figures show the serum and gastrocnemius muscle triglyceride and free fatty acid content in mice with DSS-induced colitis induced by Clostridium butyricum ML5358 in this embodiment of the invention. Figure A represents the serum triglyceride content in the control group, DSS group, and ML5358+DSS group; Figure B represents the gastrocnemius muscle triglyceride content in the control group, DSS group, and ML5358+DSS group; and Figure C represents the serum free fatty acid content in the control group, DSS group, and ML5358+DSS group. Different lowercase letters (a, b) indicate significant differences between groups. P <0.05, with the same letter indicating no significant difference between groups ( P >0.05); Figure 7 This invention illustrates the effect of Clostridium butyricum ML5358 on the expression levels of genes related to fat synthesis and uptake in mouse gastrocnemius muscle tissue. Figure 8 This invention illustrates the effect of Clostridium butyricum ML5358 on the expression levels of genes related to lipolysis and oxidation in mouse gastrocnemius muscle tissue; where different lowercase letters (a, b) indicate significant differences between groups. P <0.05, with the same letter indicating no significant difference between groups ( P >0.05). Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] Clostridium butyricum ( Clostridium butyricum ML5358 was isolated from a sample of a local Chinese pig (Guangdong Small-eared Spotted Pig) and deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37000 and deposit date of December 10, 2025.

[0026] Example 1 Isolation, culture and identification of Clostridium butyricum strain ML5358.

[0027] 1) Strain isolation and culture: Porcine intestinal contents samples were selected, serially diluted, and spread on Clostridium perfringens agar plates at different ratios. The samples were then anaerobically cultured at 37°C for 24–48 hours. Typical single colonies were selected for isolation and culture. The cultured colonies were then irradiated under a 15W UV lamp for 60, 80, and 100 seconds, respectively. After irradiation, the colonies were wrapped in aluminum foil and cultured in the dark for 48 hours. Surviving and well-growing single colonies were selected and named ML5358.

[0028] The enhanced Clostridium solid medium (RCM) consisted of: 10.0 g peptone, 10.0 g beef extract, 3.0 g yeast extract, 5.0 g glucose, 5.0 g sodium chloride, 3.0 g sodium acetate, 1.0 g soluble starch, 0.5 g L-cysteine ​​hydrochloride, and 2.0 g agar, dissolved in 1000 mL distilled water. The pH was adjusted to 6.8 ± 0.1 (25 °C), and the mixture was autoclaved at 121 °C for 15 min. After cooling to 55-60 °C, solid plates were prepared.

[0029] 2) Strain identification: Bacterial DNA was extracted from the purified strain (Sangon Biotech (Shanghai) Co., Ltd.), and PCR amplification was performed using universal bacterial 16S rRNA primers 27F and 1492R. The amplified products were sent to Sangon Sequencing Co., Ltd. in Shanghai for 16S rDNA sequencing. The obtained sequences were then compared with the NCBI database using BLAST (e.g., ...). Figure 1 As shown in the figure, Clostridium butyricum was finally obtained and named Clostridium butyricum ML5358.

[0030] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0031] 3) Morphological identification: ① Colony morphology: The selected strains were diluted with PBS and plated onto RCM solid medium. After incubation at 37°C for 24 hours, the size, color, shape, edge integrity, and transparency of the colonies were recorded. Results are as follows: Figure 2 As shown in Figure A, the colonies are milky white, opaque, with a smooth, slightly raised surface and neat edges, and a diameter of 1-2 mm.

[0032] ② Gram staining method: After smear fixation, primary staining, enzyme staining, destaining, and counterstaining, the bacterial morphology is observed under a 100° oil immersion microscope. Results are as follows: Figure 2 As shown in Figure B, Clostridium butyricum is a Gram-positive bacterium with a rod-shaped morphology.

[0033] Example 2 Growth characteristics and tolerance of Clostridium butyricum ML5358.

[0034] 1) Growth characteristics of Clostridium butyricum ML5358.

[0035] Clostridium butyricum ML5358 was inoculated at a rate of 2% (v / v) into fresh, enhanced Clostridium tumefaciens liquid culture medium and anaerobically cultured at 37°C. During the culture process, samples were taken every 2 hours, and the optical density of the culture medium at 600 nm was measured to plot a growth curve (e.g., ...). Figure 3 As shown in Figure A), the viable cell count was determined using the plate count method to plot a viable cell count curve (e.g., ...). Figure 3 As shown in Figure B), the pH value of the culture medium was measured using a pH meter to plot a pH change curve. Figure 3 (As shown in C).

[0036] The results show that Clostridium butyricum ML5358 exhibits a lag phase during the first 0-10 hours and a logarithmic growth phase during the first 10-18 hours, during which the viable cell count increases to approximately 1 × 10⁻⁶. 8 The concentration of CFU / mL initially decreased, followed by a stationary phase. After 24 hours of incubation, the pH of the culture medium decreased to approximately 4.0.

[0037] 2) Acid tolerance of Clostridium butyricum ML5358.

[0038] Adjust the pH of the sterile PBS solution to pH 2, 3, and 4 using hydrochloric acid (purchased from China National Pharmaceutical Group Co., Ltd.).

[0039] Activated *Clostridium butyricum* strain ML5358 was inoculated at a ratio of 2% into PBS solutions of different pH values ​​and cultured at 37°C. Samples were taken every 1 hour for plating, with three replicates for each treatment. The viable count of *Clostridium butyricum* ML5358 after different tolerance times was calculated using the plate count method. Finally, the viable count at 0 h was used as a control to calculate the survival rate of *Clostridium butyricum* ML5358 under different acidic conditions for different durations (e.g., 0 h viable count). Figure 4 (As shown in A).

[0040] The results show that Clostridium butyricum ML5358 exhibits excellent stability under acidic conditions. After exposure to PBS solutions at pH 2, 3, and 4 for 3 hours, its survival rates remained at 3.97%, 15.26%, and 45.05%, respectively.

[0041] 3) Bile salt tolerance of Clostridium butyricum ML5358.

[0042] Solutions containing 0%, 0.1%, 0.2%, and 0.3% bile salts (purchased from Solarbio, China) were prepared using sterile PBS. These solutions were then inoculated at a 2% concentration with activated *Clostridium butyricum* strain ML5358 and incubated at 37°C. Samples were taken at 1-hour intervals for plating, with three replicates per treatment. The number of viable bacteria tolerating different time points was calculated using the plate count method. Finally, using the number of viable bacteria at a concentration of 0% as a control, the survival rate of *Clostridium butyricum* ML5358 under different bile salt conditions was calculated for different time points. (e.g.) Figure 4 (As shown in B).

[0043] The results show that Clostridium butyricum ML5358 exhibits significant bile salt tolerance. Even after 3 hours of exposure to a high concentration of 0.2% bile salts, its survival rate remained at 2.55%.

[0044] Example 3 Clostridium butyricum ML5358 alleviates colitis in DSS mice.

[0045] Twenty-four 10-week-old male BALB / c rats were randomly divided into three groups of eight each.

[0046] The first phase of the experiment lasted 6 days. The control group and the DSS group were administered normal saline by gavage, while the Clostridium butyricum ML5358+DSS group was administered Clostridium butyricum ML5358 bacterial suspension (1×10⁻⁶) by gavage every other day. 8 200 μL of CFU / mL bacterial suspension.

[0047] In the second stage, based on the first stage, 1.5% DSS was added to the drinking water of mice in the DSS group and the Clostridium butyricum group for 14 days. During the experiment, the temperature and humidity in the mouse housing were kept constant, and the mice were allowed free access to food and water. The survival status of the mice was observed and recorded throughout the experiment. On the last day of the experiment, the mice were euthanized by cervical dislocation, and their serum, muscle tissue, and colon tissue were collected.

[0048] RNA was extracted from tissues using the Trizol method and its concentration was determined. After reverse transcription, the expression levels of inflammatory factors (Il-6, Il-1β, and Tnf-α) were detected by real-time quantitative PCR. Primers used are shown in Table 1.

[0049] Table 1 Primers for detecting inflammatory factors in mice

[0050] The results are as follows Figure 5 As shown, the Clostridium butyricum ML5358+DSS group significantly reduced the expression levels of Il-1β and Il-6 inflammatory factors in the mouse colon, and had the ability to alleviate DSS-induced colitis.

[0051] Example 4 Clostridium butyricum ML5358 increases intramuscular fat content in mice.

[0052] Building upon Example 3, we further investigated the triglyceride and free fatty acid content in the serum and gastrocnemius muscle of Clostridium butyricum ML5358 mice using a triglyceride and free fatty acid assay kit (purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.). The triglyceride detection employed the GPO-PAP method. In this method, serum triglycerides are hydrolyzed into glycerol and free fatty acids by lipoprotein lipase, followed by glycerol phosphorylation and oxidation. This process generates hydrogen peroxide, which reacts with the chromogenic reagent. The content is calculated by measuring the absorbance and comparing it with a standard. The free fatty acid assay kit operates on a similar principle.

[0053] Simultaneously, we extracted RNA from gastrocnemius muscle tissue using the Trizol method, detected its concentration, and then performed reverse transcription. Real-time quantitative PCR was then used to detect the expression levels of lipogenesis genes (Fasn, Srebf1, and Acc1) and lipolysis genes (Atgl, Pparα, Cpt1α, Cd36, Lpl, Fabp4, and Pgc1α). Primers used are shown in Table 2.

[0054] Table 2 Primers for amplification of mouse fat synthesis and breakdown genes

[0055] like Figure 6 Results A and B showed that, compared with the control group, the Clostridium butyricum ML5358+DSS group significantly increased the triglyceride content in the serum and gastrocnemius muscle of mice. Figure 6 The results showed no significant difference in serum free fatty acid content. Figures 7-8 As shown, among the genes promoting fat synthesis and uptake (Fasn, Srebf1, Acc1, Fabp4 and Cd36), the expression levels of Srebf1, Acc1, and Fabp4 were significantly increased in the Clostridium butyricum ML5358+DSS group, while the expression levels of genes related to fat breakdown and oxidation (Atgl, Pparα, Cpt1b, Lpl, and Pgc1α) showed no difference.

[0056] In summary, Clostridium butyricum ML5358 has the ability to promote intramuscular fat production, thereby promoting lipid synthesis and reducing lipid breakdown.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Clostridium butyricum ML5358, characterized in that, The Clostridium butyricum ML5358 was deposited on December 10, 2025, with accession number CGMCC No. 37000.

2. A microbial agent, characterized in that, The microbial agent includes Clostridium butyricum ML5358 as described in claim 1 or the fermentation product of Clostridium butyricum ML5358.

3. A feed additive, characterized in that, The feed additive includes Clostridium butyricum ML5358 as described in claim 1 or the inoculant as described in claim 2.

4. The feed additive according to claim 3, characterized in that, The viable count of Clostridium butyricum ML5358 in the feed additive is not less than 1 × 10⁻⁶ per unit weight or volume. 8 CFU / mL or 1×10 8 CFU / g.

5. The use of the feed additive according to any one of claims 3 to 4 in the preparation of products for increasing intramuscular fat content in animals.

6. The use of the feed additive according to any one of claims 3 to 4 in the preparation of products for the prevention and / or relief of intestinal inflammation in animals.

7. The application according to claim 6, characterized in that, The product can reduce the expression levels of inflammatory factors Il-6 and Il-1β in colon tissue.

8. The use of the feed additive according to any one of claims 3 to 4 in the preparation of products for regulating lipid metabolism in animal skeletal muscle.

9. The application according to claim 8, characterized in that, The regulation of skeletal muscle lipid metabolism includes promoting the expression of genes related to fat synthesis and uptake.

10. The application according to claim 9, characterized in that, The genes related to fat synthesis and uptake include at least one of Srebf1, Acc1, and Fapp4.

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