Lactobacillus reuteri and its use in promoting slow muscle fibers in muscle

Abstract

This invention belongs to the field of microbial applications, specifically relating to *Lactobacillus reuteri* and its application in promoting slow-twitch muscle fibers. The *Lactobacillus reuteri*, with accession number CGMCC No. 36844, was deposited at the China General Microbiological Culture Collection Center on December 3, 2025. This invention is the first to use *Lactobacillus reuteri* M3 to feed Mongolian horses, and then performs immunofluorescence and RT-qPCR detection on the horse muscles. Immunofluorescence images visually demonstrate the increase in the proportion of slow-twitch muscle fibers in the horses after feeding *Lactobacillus reuteri* M3. This invention clarifies through PCR detection that *Lactobacillus reuteri* M3 can drive muscle fiber remodeling towards a slow-twitch phenotype at the transcriptional level, providing scientific evidence for the product's efficacy.

Description

Technical Field

[0001] This invention belongs to the field of microbial applications, specifically relating to a type of *Lactobacillus reuteri* and its application in promoting slow-twitch muscle fibers. Background Technology

[0002] In competitive sports and healthy aging, skeletal muscle function is crucial. The composition of skeletal muscle fiber types is closely related to athletic performance; endurance athletes have a high proportion of slow-twitch muscle fibers, while explosive athletes are predominantly fast-twitch fibers. A high proportion of slow-twitch muscle fibers is generally associated with excellent endurance performance, fatigue resistance, and energy metabolism homeostasis. Multiple population studies have confirmed that the higher the proportion of slow-twitch muscle fibers, the better the athlete's tolerance to long-distance exercise. This finding suggests that increasing the proportion of slow-twitch fibers may improve endurance performance. For endurance racehorses, the proportion of slow-twitch muscle fibers is a key indicator distinguishing "excellent riders" from "average riders." A classic 1996 study found that horses with excellent endurance race results had significantly higher proportions of Type I (slow-twitch) and Type IIA (oxidative fast-twitch) fibers than horses with average results, while having significantly lower proportions of Type IIB (glycolytic fast-twitch) fibers. Fiber type data from muscle biopsies can effectively identify a horse's endurance potential.

[0003] Currently, known methods for regulating myofiber type conversion mainly include long-term endurance training, drug interventions such as PPARδ agonists, and gene interventions such as overexpression of PGC-1α or silencing NFAT via AAV viral vectors. However, equine training cycles are extremely long, typically requiring months to years of sustained high-intensity training to induce substantial changes in myofiber type, which does not meet the time-sensitive requirements of racehorse training. Training effectiveness is highly dependent on the experience level of jockeys and trainers, and different horses respond significantly differently to the same training program, lacking stability and predictability. PPARδ agonists (such as GW501516) have been explicitly listed as prohibited substances by the International Federation of Horseracing Habitats (IFHA) and the World Anti-Doping Agency (WADA), and their use in equestrian competitions is a violation, resulting in suspension and penalties upon detection. Animal studies with long-term use of such drugs have shown a risk of multi-organ cancers (such as liver and intestinal cancer), posing an unacceptable risk to the health and welfare of horses. The application of gene therapy in horses faces strict ethical reviews and regulatory restrictions. Most horse racing management agencies and equestrian organizations explicitly prohibit gene editing or gene therapy aimed at enhancing performance. Furthermore, gene manipulation is technically demanding and extremely expensive, making it unsuitable for widespread adoption as a routine equine management method. Therefore, developing a novel strategy to regulate equine muscle fiber types, addressing the shortcomings of existing technologies, has significant practical implications and promising application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a *Lactobacillus reuteri* that solves the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: This invention provides a strain of *Lactobacillus reuteri* that increases the proportion of slow-twitch muscle fibers. The *Lactobacillus reuteri* strain has the accession number CGMCC No. 36844 and was deposited at the China General Microbiological Culture Collection Center on December 3, 2025.

[0006] A second aspect of the present invention provides a fermentation broth of *Lactobacillus reuteri*, the fermentation broth being derived from the fermentation of *Lactobacillus reuteri*.

[0007] A third aspect of the present invention provides a method for preparing the fermentation broth, comprising the following steps: Seed culture was obtained by culturing *Lactobacillus reuteri* in MRS medium at 35℃~38℃ for 18h~30h. The seed culture was inoculated into MRS medium and cultured at 35℃~38℃ for 18h~30h to expand the culture, thus obtaining the fermentation broth.

[0008] Preferably, the conditions for preparing the seed culture are: incubation at 37°C for 24 hours; The conditions for expanded culture were 37℃ for 24 hours.

[0009] Preferably, the inoculation volume of the seed culture is 2% of the volume of the MRS medium.

[0010] The fourth aspect of the present invention provides an application of the aforementioned *Lactobacillus reuteri* and / or the aforementioned fermentation broth, wherein the application refers to the preparation of microbial agents to increase the proportion of slow-twitch muscle fibers in horses.

[0011] Preferably, the preparation method of the microbial agent is as follows: The fermentation broth was centrifuged at 8000 rpm for 10 min, and the precipitate was collected.

[0012] Adjust the concentration of the precipitate to 1×10 using water. 10 CFU / mL ~5×10 13 The microbial agent is obtained by measuring CFU / mL.

[0013] Preferably, the viable count in the microbial agent is 1×10⁻⁶. 13 CFU / mL.

[0014] Preferably, the horse is a Mongolian horse.

[0015] The basic information of *Lactobacillus reuteri* described in this invention is as follows: M3, the suggested classification name is *Lactobacillus reuteri*.Limosilactobacillus reuteri The accession number is CGMCC No.36844. It was deposited on December 3, 2025 at the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a *Lactobacillus reuteri* strain that promotes slow-twitch muscle fiber development. The *Lactobacillus reuteri* strain, with accession number CGMCC No. 36844, was deposited at the China General Microbiological Culture Collection Center on December 3, 2025. This invention is the first to use *Lactobacillus reuteri* M3 to feed Mongolian horses, and then performs immunofluorescence and RT-qPCR detection on the horse muscles. Immunofluorescence images clearly demonstrate the increase in the proportion of slow-twitch muscle fibers in the horses after feeding *Lactobacillus reuteri* M3, providing strong evidence. This invention clarifies through PCR detection that *Lactobacillus reuteri* M3 can drive muscle fiber remodeling towards a slow-twitch phenotype at the transcriptional level, providing scientific support for the product's efficacy. Compared to gene therapy and drugs, oral lactic acid bacteria are highly safe microecological preparations that can be used long-term as food or feed additives. Attached Figure Description

[0017] Figure 1 Phylogenetic analysis of *Lactobacillus reuteri*.

[0018] Figure 2 Immunofluorescence results of fast and slow muscle fibers after feeding with *Lactobacillus reuteri*; A: Representative images of immunofluorescence from three replicates in different groups. Green fluorescence represents fast-twitch muscle fibers, red fluorescence represents slow-twitch muscle fibers, and blue represents cell nuclei. B: Statistical results from Figure A. Blue represents the percentage of fast-twitch muscle fibers, and red represents the percentage of slow-twitch muscle fibers. *P<0.05. ***P<0.001.

[0019] Figure 3 The values ​​represent the mRNA expression levels of genes related to slow-twitch and fast-twitch muscle fibers in different groups. *P<0.05. **P<0.01. ***P<0.001. Detailed Implementation

[0020] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0021] The inventive concept of this invention is as follows: This invention is based on the theory of the "gut-muscle axis," which states that gut microbiota and their metabolites can influence the metabolic state and gene expression profile of distal skeletal muscle through blood circulation, immune regulation, and neuroendocrine pathways, thus providing a scientific basis for regulating muscle fiber type through oral probiotics. Guided by this theory, the inventors selectively isolated, purified, and identified a strain of *Lactobacillus reuteri* (equine-derived) from the feces of Mongolian endurance racehorses. Limosilactobacillus reuteri M3, with accession number CGMCC No. 36844. This strain is derived from the same animal host, exhibiting good host adaptability and biosafety, avoiding potential problems such as immune rejection or intestinal colonization difficulties caused by exogenous strains. Furthermore, this invention, through a rigorously designed animal control experiment, administered low-dose (2 g / day) and high-dose (5 g / day) intragastric interventions to Mongolian horses for 6 consecutive weeks. Combining histological immunofluorescence staining and RT-qPCR molecular detection, this invention, for the first time, demonstrated in live horses that oral administration of this strain significantly increases the proportion of slow-twitch muscle fibers (type I) in the gluteus medius muscle, and at the transcriptional level, upregulates the expression of slow-twitch muscle marker genes (TNNT1, TNNC1, PERM1, PGC1α, CPT1B) and downregulates the expression of fast-twitch muscle marker gene (MYLPF), thus forming a closed chain of evidence at both the phenotypic and molecular levels. This invention not only avoids the safety risks and compliance issues of drugs and gene therapy, but also provides a feed additive or microbial agent solution that is easy to operate, cost-controllable, and scalable. It is especially suitable for horse racing training, daily management of endurance sports horses, and muscle function maintenance of old or rehabilitated horses, and has significant industrial application value and animal welfare significance.

[0022] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0023] Example 1 A type of *Lactobacillus reuteri*, specifically as follows: In this invention, 5 g of collected Mongolian Masai horse feces was weighed into a 50 mL centrifuge tube, 45 mL of PBS was added, and the mixture was vortexed. The mixture was then diluted using a gradient dilution method, and 10 g of the diluted feces was selected. -4 10 -5 10 -6Three dilutions were prepared, with 100 μL of each diluted solution spread onto pre-prepared MRS agar plates and incubated anaerobicly at 37°C for 48 h. Uniform, well-defined single colonies were picked from the MRS agar plates, numbered, and inoculated into MRS liquid medium for subculturing. After stable proliferation, the strain was purified using the streak plating method. The purified strain was then incubated anaerobically at 37°C for 24 h in MRS liquid medium.

[0024] DNA was extracted from the cultured bacterial strain according to the instructions accompanying the bacterial genomic DNA extraction kit (DP302-02) provided by Tiangen Biotech (Beijing) Co., Ltd. PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rRNA gene; the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The PCR reaction system and conditions are shown in Tables 1 and 2.

[0025] SEQ ID NO. 1: 5'-AGAGTTTTGATCCTGGCTCA-3'.

[0026] SEQ ID NO. 2: 5'-GGTTACCTTGTTACGACTT-3'.

[0027] Table 1 PCR reaction system Table 2 PCR reaction conditions PCR amplification products were detected by 1.2% agarose gel electrophoresis, and the results were observed using a gel imaging system. Using 27F and 1492R as sequencing primers, the PCR amplification products were sent to Beijing BGI Genomics Co., Ltd. for sequencing.

[0028] The sequence data obtained from sequencing were compared with the NCBI (National Center for Biotechnology Information) database using BLAST to obtain homology sequences. A phylogenetic tree was then constructed using MEGA11 software to clarify the strain's attributes. The sequencing results of the isolated strain's 16S rRNA are shown in SEQ ID NO.3 below.

[0029] SEQ ID NO.3:

[0030] Based on such Figure 1 The phylogenetic tree constructed from the 16S rRNA gene shown shows that the bacteria isolated in this invention belong to the same species as *Lactobacillus reuteri*, and are named *Lactobacillus reuteri* M3.

[0031] Example 2 The application of *Lactobacillus reuteri* in promoting slow-twitch muscle fibers is as follows: 1. Materials and Methods: Lactobacillus reuteri M3 strain was cultured in MRS liquid medium at 37°C for 24 h anaerobic culture to obtain seed culture. The seed culture was then transferred to sterilized MRS liquid medium at an inoculum rate of 2% (v / v) and cultured at 37°C for 24 h. The culture was centrifuged at 8000 rpm for 10 min, and the precipitate was collected to obtain Lactobacillus reuteri M3 bacterial sludge. The sludge was resuspended twice with distilled water and collected, and its weight was measured. 2 g and 5 g of the sludge were resuspended in 500 mL of water respectively, adjusting the concentration to 10. 13 CFU / mL, in preparation for subsequent horse feeding trials.

[0032] Healthy Mongolian stallions, approximately 3 years old and weighing 300 kg, were selected as experimental animals at the Xiwuzhumuqin White Horse Base. Based on the equivalent dose ratio table for humans and animals calculated by body surface area in the "Pharmacology Experiment" textbook, the dosage of *Lactobacillus reuteri* was determined through dual calculations based on the horses' weight and body surface area. The experiment included three groups: a control group (equal volume of water), a low-dose (2g) group, and a high-dose (5g) group, with 3 horses in each group. To prevent variations in intake per horse and to avoid waste, the bacterial sludge was dissolved in water and administered to the horses via gavage, ensuring complete drug entry into the horses' bodies. Gavage was performed daily for 6 consecutive weeks.

[0033] Following gavage, the horses were sedated and analgesicated according to animal welfare principles, using butorphanol combined with detopromidine hydrochloride for anesthesia. A biopsy was then performed on the gluteus medius muscle. This muscle is a commonly used sampling site for assessing equine skeletal muscle function and adaptive changes, characterized by stable muscle fiber composition and high reproducibility, reliably reflecting movement-related muscle physiological changes. To minimize individual variability and technical bias, all samples were collected from the same anatomical location and sampling depth. The collected muscle tissue samples were divided into two parts: one part was immediately and rapidly frozen in liquid nitrogen for subsequent nucleic acid extraction, and RT-qPCR was used for quantitative detection of fast-twitch and slow-twitch muscle marker genes; the other part was fixed in 4% paraformaldehyde solution for histological observation and immunostaining analysis.

[0034] Skeletal muscle immunofluorescence staining: Muscle samples fixed in 4% paraformaldehyde solution were paraffin-embedded and sectioned. Immunofluorescence double staining was performed using fast-muscle antibody (mouse anti, Abcam China) and slow-muscle antibody (rabbit anti, Abcam China). The proportion of fast and slow muscle fibers was calculated using Image-View software.

[0035] The methods for extracting and analyzing total RNA from skeletal muscle are as follows: (1) Skeletal muscle samples need to be taken out from -80℃ and ground into powder.

[0036] (2) Add 1 ml of Trizol to every 100 mg of skeletal muscle sample powder, shake thoroughly to mix, and let stand for 30 min.

[0037] (3) Centrifuge at low temperature and high speed for 10 min (4℃, 12000 rpm), transfer the supernatant to a new tube and add chloroform (200 µl of chloroform per 1 ml Trizol) and mix well.

[0038] (4) Centrifuge at low temperature and high speed for 10 min (4℃, 12000 rpm). The liquid is divided into three phases. The upper colorless liquid phase is transferred to a new tube and 500 µl of isopropanol is added and mixed.

[0039] (5) Centrifuge at low temperature and high speed for 10 min (4℃, 12000 rpm), discard the supernatant and add 1 ml of ethanol (75%) to elute.

[0040] (6) Centrifuge at low temperature and high speed for 5 min (4℃, 12000 rpm), discard the supernatant and add 1 ml of ethanol (75%) to elute again.

[0041] (7) After the ethanol evaporates, add 30 μL of sterile, enzyme-free water to dissolve the RNA precipitate according to the size of the precipitate, and store it in an ultra-low temperature freezer at -80℃ for later use.

[0042] (8) Total RNA quality control: RNA purity and concentration were detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0043] (9) RNA was reverse transcribed into cDNA using Prime Script TMRT Master Mix (RR036A, Takara). The following liquid was added to a 200 μL sterile enzyme-free tube (the reaction solution was prepared on ice). The reverse transcription process was strictly performed according to the kit instructions.

[0044] The reaction system consisted of 10 μL of 5× PrimeScript RT Master Mix 2 μL, with a maximum total RNA volume of 500 ng, and RNase-free ddH2O added to 10 μL.

[0045] The reverse transcription procedure is as follows: 37 ℃ for 15 min, 85 ℃ for 5 s, cool down to 4 ℃ to terminate the reaction, and store at -20 ℃ for later use.

[0046] The steps for real-time quantitative PCR are as follows: (1) Prepare the reaction solution with the reverse transcribed cDNA according to the instructions of TaKaRa's SYBR Rremix Ex Taq™ II reagent.

[0047] (2) Reaction system: 10 μL of TB Green Premix Ex Taq II (2×), 0.8 μL of PCR Forward Primer, 0.8 μL of PCR Reverse Primer, 0.4 μL of Rox Reference Dye II, 2 μL of DNA template (<100 ng), and RNase free water to make up to 20 μL.

[0048] (3) Place it in a real-time PCR instrument for reaction. The reaction process is 95℃, 30s; 95℃, 5s, 60℃, 30s, 95℃, 15s, 40 cycles; 60℃, 1min; 95℃, 15s.

[0049] (4) Using 2 -ΔΔCt The method is used for analysis.

[0050] 2. Results.

[0051] Immunofluorescence double staining was performed on the gluteus medius muscle of nine horses. Figure 2 The immunofluorescence results of gluteus medius muscle fiber types in 9 horses are shown. Immunofluorescence results of the control group, low-dose group (2g), and high-dose group (5g) showed that the expression of slow muscle fibers (red) was significantly higher in the control group after feeding 2g and 5g of Lactobacillus reuteri M3 (P<0.01).

[0052] Further analysis of all images was conducted using Image-View software to calculate the specific proportions of fast and slow muscle fibers in the gluteus medius muscle of nine Mongolian horses after feeding them with Lactobacillus reuteri M3. Statistical analysis was performed on the results, and corresponding bar charts were generated. Figure 2 As shown in Figure B, statistical results indicated that the low-dose group fed with *Lactobacillus reuteri* M3 had the highest proportion of slow-twitch muscle fibers. Compared with the control group, both low-dose and high-dose feeding with *Lactobacillus reuteri* M3 significantly increased the proportion of slow-twitch muscle fibers (P<0.01); the proportion of slow-twitch muscle fibers in the low-dose group was significantly higher than that in the high-dose (H) group (P<0.05). Immunofluorescence results suggested that oral administration of *Lactobacillus reuteri* M3 effectively promoted the expression of slow-twitch muscle fibers in the gluteal medius muscle of Mongolian horses.

[0053] To further verify the molecular-level regulatory characteristics of myofiber type changes, this invention detected the mRNA expression levels of typical fast-twitch muscle fibers (TNNI2 and MYLPF) and slow-twitch muscle fibers (TNNT1, TNNC1, PERM1, PGC1α, and CPT1B). The results are as follows: Figure 3 The results showed that in the 2 g and 5 g *Lactobacillus reuteri* M3 treatment groups, the mRNA expression levels of slow muscle-related genes TNNT1, TNNC1, PERM1, PGC1α, and CPT1B were significantly upregulated (P<0.05), while the mRNA expression level of fast muscle-related gene MYLPF was significantly downregulated (P<0.05), suggesting that *Lactobacillus reuteri* M3 can drive myofibrillary remodeling towards a slow muscle phenotype at the transcriptional level.

[0054] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A type of *Lactobacillus reuteri* that increases the proportion of slow-twitch muscle fibers, characterized in that, The Lactobacillus reuteri described herein has the accession number CGMCC No. 36844 and was deposited at the China General Microbiological Culture Collection Center on December 3, 2025.

2. A fermentation broth of *Lactobacillus reuteri*, characterized in that, The fermentation broth is derived from Lactobacillus reuteri as described in claim 1.

3. The method for preparing fermentation broth as described in claim 2, characterized in that, The steps are as follows: Seed culture was obtained by culturing *Lactobacillus reuteri* in MRS medium at 35℃~38℃ for 18h~30h. The seed culture was inoculated into MRS medium and cultured at 35℃~38℃ for 18h~30h to expand the culture, thus obtaining the fermentation broth.

4. The preparation method according to claim 3, characterized in that, The conditions for preparing the seed culture were: incubation at 37℃ for 24 hours; The conditions for expanded culture were 37℃ for 24 hours.

5. The preparation method according to claim 3, characterized in that, The seed culture was inoculated at 2% of the volume of the MRS medium.

6. The application of *Lactobacillus reuteri* as described in claim 1 and / or the fermentation broth as described in claim 2, characterized in that, The application refers to the preparation of microbial agents to increase the proportion of slow-twitch muscle fibers in horses.

7. The application as described in claim 6, characterized in that, The preparation method of the microbial inoculant is as follows: The fermentation broth was centrifuged at 8000 rpm for 10 min, and the precipitate was collected. Adjust the concentration to 1×10 using water. 10 CFU / mL ~5×10 13 The microbial agent is obtained by measuring CFU / mL.

8. The application as described in claim 7, characterized in that, The viable bacteria count in the microbial agent is 1×10⁻⁶. 13 CFU / mL.

9. The application as described in claim 6, characterized in that, The horse in question is a Mongolian horse.

Images