Synthetic microbial communities syncoms-r for ruminants and uses thereof

Abstract

The present application belongs to the technical field of animal digestive tract health and function regulation, and particularly relates to a synthetic microbial community SynComs-R for ruminants and application thereof. The community is composed of Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus rhamnosus MRY6 and Enterococcus faecalis G1. The four strains are functionally complementary and metabolically synergistic, forming an integrated microbial function chain. The present application can realize bidirectional "correction and optimization" regulation across species and physiological states, and has the dual-site precise regulation effect of reducing redundancy in the rumen and promoting digestion, and increasing diversity in the intestinal tract and strengthening immunity. The present application can significantly improve the production performance and nutrient digestibility of ruminants, enriches butyric acid-producing beneficial bacteria and inhibits pathogenic bacteria, and has a significant anti-inflammatory effect through metabolic-immune synergy. The present application can be prepared into a microbial agent or a feed additive and applied to ruminant health breeding.

Description

Technical Field

[0001] This invention belongs to the field of animal digestive tract health and function regulation technology, specifically relating to a synthetic microbial community SynComs-R for ruminants and its applications. Background Technology

[0002] Ruminants rely on the highly complex and dynamically balanced microbial community within their rumen to efficiently convert and utilize roughage resources, with rumen fermentation being one of their core physiological characteristics. Bacteria, archaea, fungi, and other microorganisms colonizing the rumen gradually degrade macromolecular nutrients such as fiber, starch, and protein in the diet through symbiotic metabolism, ultimately converting them into key metabolites such as volatile fatty acids (VFAs) and microbial protein (MCP). VFAs can provide 70%–75% of the host's metabolizable energy, while MCPs can meet 40%–98% of the host's daily protein requirements. Therefore, rumen fermentation efficiency directly determines the feed conversion ratio and overall production performance of ruminants. Simultaneously, the gut, as the primary site of nutrient absorption and a crucial barrier for the body's immune defense, exhibits a close synergistic regulatory relationship between its microbial community structure and health status and rumen fermentation function. Both factors jointly influence the animal's overall metabolic level and immune function.

[0003] Probiotics, as a class of live microorganisms that can produce beneficial effects on the host, can improve animal health and production performance through various pathways, such as regulating the structure of the digestive tract flora, enhancing host immune function, and promoting the digestion and absorption of nutrients. They are an important technological solution for replacing antibiotics in current green ruminant farming, and their application in large-scale ruminant breeding is becoming increasingly widespread. However, current ruminant probiotic products on the market still have many technical limitations, making it difficult to fully meet the precise control needs of different farming scenarios. Specifically, these limitations are reflected in the following aspects: First, their functional scenarios are fixed, lacking broad-spectrum applicability across physiological states and species. Current probiotic preparations are generally designed for single scenarios such as "health promotion and growth enhancement" or "stress repair," meaning they only function under specific physiological conditions and cannot simultaneously achieve the dual regulatory function of "correcting and repairing abnormal physiological conditions and enhancing efficacy and promoting optimization under normal physiological conditions." Furthermore, the strain screening and efficacy verification of most products are only for a single ruminant species, resulting in insufficient compatibility and efficacy stability across different ruminant species. The lack of probiotic products with broad-spectrum cross-species application potential limits their application scenarios and makes it difficult to cover the diverse needs of aquaculture production.

[0004] Second, the regulation of the entire digestive tract lacks site-specific precision and fails to take into account the ecological functional differences between the rumen and the gut. The rumen and gut differ significantly in their physiological environment, microbial community structure, and core physiological functions: the rumen's core functions are fermentation and degradation of substrates and synthesis of metabolites, requiring improvements in fermentation efficiency through reducing microbial redundancy and targeted enrichment of core functional microorganisms; the gut's core functions are nutrient absorption and immune defense, requiring enhancement of microbial diversity and enrichment of beneficial microorganisms to strengthen the intestinal barrier function. Existing products mostly adopt a homogeneous regulatory approach, failing to design differentiated regulatory strategies based on the different ecological characteristics of the rumen and gut. This makes it difficult to simultaneously achieve precise regulation of both "reducing rumen redundancy and promoting digestion" and "enhancing gut diversity and strengthening immunity," thus failing to fully leverage the efficacy of synergistic regulation across the entire digestive tract.

[0005] Third, metabolic regulation and anti-inflammatory immune effects are separated, and the functional synergy and mechanisms of action among strains are unclear. Existing compound probiotic products are mostly based on the simple superposition of single-strain functions, lacking a systematic design based on the laws of microbial ecological interactions and metabolic pathway connections. The synergy between strains in substrate utilization and metabolite interactions is insufficient, making it difficult to produce a super-synergistic effect. Furthermore, most products only focus on nutrient metabolism regulation or antibacterial and anti-inflammatory effects, failing to systematically couple "energy metabolism remodeling" with "immune pathway inhibition." They lack integrated design and systematic experimental verification of multi-dimensional functions such as oxygen consumption, enzyme production, acid production, and anti-inflammation, resulting in unclear product mechanisms of action and poor stability and reproducibility of effects in practical applications.

[0006] The aforementioned technical deficiencies are common problems in the current field of ruminant microecological preparations, directly resulting in limited efficacy, poor batch stability, and weak species universality of existing probiotic products in actual livestock production. This severely restricts the promotion and application of microecological preparations in the healthy breeding of ruminants. Currently, there is a lack of synthetic microbial community products and targeted solutions in this field that can simultaneously achieve bidirectional regulation across physiological states, precise division of labor at two sites in the rumen and gut, and synergistic effects of metabolism and immunity. Summary of the Invention

[0007] The purpose of this invention is to provide a synthetic microbial community SynComs-R for ruminants and its application, in which the strains complement each other and work synergistically to ensure the overall effectiveness of the synthetic community.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides a synthetic microbial community SynComs-R for ruminants, composed of four functionally complementary strains: Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1. The preservation numbers for Bacillus licheniformis MY176, YCNL1, MRY6, and G1 are: CCTCC NO: M 2026425, CGMCC NO: 29050, MRY6, and MRY6 respectively.

[0009] Furthermore, the viable count ratio of Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus reuteri MRY6 and Enterococcus faecalis G1 is (0.5~1.5):(0.5~1.5):(0.5~1.5):(0.5~1.5).

[0010] The present invention also provides a microbial inoculant comprising the aforementioned synthetic microbial community SynComs-R.

[0011] Furthermore, the microbial agent is in the form of a liquid agent, a freeze-dried powder, or microcapsule granules; the total viable count in the liquid agent is not less than 1 × 10⁻⁶. 8 CFU / mL.

[0012] The present invention also provides a ruminant feed additive comprising the synthetic microbial community SynComs-R or the microbial agent described above.

[0013] Furthermore, this also includes feed-grade acceptable additives.

[0014] The present invention also provides the application of the aforementioned synthetic microbial community SynComs-R in the preparation of products for optimizing rumen fermentation in ruminants and promoting intestinal health.

[0015] Furthermore, the optimization of rumen fermentation and promotion of intestinal health in ruminants specifically includes: achieving bidirectional regulation across physiological states, correcting intestinal flora imbalance and restoring the flora to normal physiological levels under abnormal stress conditions, and improving animal production performance and nutrient digestion efficiency under normal physiological conditions; achieving differentiated regulation at two sites in the rumen and gut, reducing rumen flora redundancy, selectively enriching fiber-degrading core flora to improve fermentation efficiency, increasing intestinal flora diversity, enriching butyrate-producing beneficial bacteria and inhibiting pathogenic bacteria to enhance the intestinal immune barrier; increasing fiber degradation rate and bacterial protein synthesis, selectively regulating volatile fatty acid composition to increase the proportion of propionic and butyrate, autonomously stabilizing rumen pH, inhibiting the proliferation of harmful intestinal bacteria, enhancing intestinal barrier function, and exerting systemic anti-inflammatory effects.

[0016] Furthermore, the product is a ruminant feed additive.

[0017] Furthermore, the ruminants include dairy cows, sheep, and cattle.

[0018] The beneficial effects of this invention are as follows: This invention can rapidly correct gastrointestinal flora imbalance in dairy cows under abnormal physiological stress during the peripartum period, promoting the rapid recovery of the flora structure to peak lactation levels and significantly alleviating the decline in production performance caused by peripartum stress. It increases the average daily milk yield of dairy cows in the peripartum period (3-108 days postpartum) by 10% compared to the control group. Under normal physiological conditions in sheep and cattle, it can further enhance digestive and metabolic functions and improve production performance. Specifically, in sheep, the average daily weight gain increased by 20.5% and dry matter digestibility by 7.4% in days 28-56; in cattle, the average daily weight gain increased by 33.6% and dry matter digestibility by 27.8% in days 30. Simultaneously, it reduces the feed conversion ratio, improving the economic benefits of animal husbandry. This synthetic community exhibits a stable and consistent positive regulatory effect in all three ruminants—dairy cows, sheep, and cattle—and is not limited by species or physiological stage, demonstrating high universality and application stability.

[0019] The SynComs-R synthetic microbial community of this invention can reduce the redundancy of the rumen microbiota, selectively enrich the core fiber-degrading microbiota such as Prevotella, and optimize the functional structure of the rumen microbiota to improve fermentation efficiency. Validation data show that the degradation rate of neutral detergent fiber in periparturient dairy cows increased by 12.19%, and the synthesis of microbial protein in meat sheep increased by 13.82%. In in vitro rumen fermentation experiments, the SynComs-R group showed a 15.6% increase in gas production and a 10.34% increase in microbial protein production at 48 hours compared to the control group. All fermentation indicators were significantly better than those of the single-strain treatment groups. At the intestinal level, this community enhances microbial diversity, selectively enriching butyrate-producing beneficial bacteria such as UCG-005 and Agathobacter, while inhibiting the proliferation of potentially pathogenic bacteria such as Escherichia-Shigella. Specifically, in peripartum dairy cows, the abundance of UCG-005 increased by 28.97%, Agathobacter by 85.06%, and the abundance of pathogenic Escherichia-Shigella decreased by 97.89%. In meat sheep, the abundance of butyrate-producing beneficial bacteria also significantly increased, effectively strengthening the intestinal immune barrier function. The rumen enhances nutrient degradation and conversion efficiency, while the intestinal level strengthens nutrient absorption and immune defense; this dual-site synergistic effect achieves overall optimization of the entire digestive tract function.

[0020] At the metabolic level, this invention can significantly upregulate rumen glycolysis / gluconeogenesis and pyruvate metabolism pathways, enhance the succinate pathway and butyrate kinase pathway, and directionally optimize the composition of volatile fatty acids. The total volatile fatty acids in the rumen of peripartum dairy cows are increased by 12.77%, of which butyrate is increased by 49.77% and propionic acid content is significantly increased. The butyrate content in the intestine is increased simultaneously, providing efficient energy substrates for intestinal epithelial cells and further enhancing intestinal barrier function. At the immune level, this community exerts a systemic anti-inflammatory effect by reducing lipopolysaccharide release and weakening TLR4 pathway activation through the reduction of Gram-negative bacteria, and by inhibiting the NF-κB inflammatory signaling pathway through the enriched butyrate, which acts as an HDAC inhibitor. In peripartum dairy cow trials, compared with the recognized highly effective microbiota transplantation technology, the SynComs-R group showed a 7.08% reduction in the pro-inflammatory factor TNF-α and an 8.83% increase in the anti-inflammatory factor IL-10, demonstrating a more comprehensive and superior anti-inflammatory effect. In sheep trials, serum IL-6, TNF-α, and IL-1β decreased by 56.5%, 47.0%, and 50.9%, respectively, showing a significant anti-inflammatory effect. This metabolic-immune synergistic mechanism is clear and well-defined, providing solid theoretical support for the stability and reproducibility of the product's efficacy.

[0021] The synthetic microbial community SynComs-R of this invention consists of four strains: Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1. These four strains form a complete functional loop in substrate utilization and product interaction, with their metabolites serving as substrates for each other, constructing an indispensable microbial functional chain rather than a simple superposition of single-strain functions. The four strains were obtained from a strain library containing 172 candidate probiotics through a large-scale antagonistic screening of 14,706 combinations and multiple rounds of in vitro rumen fermentation rescreening. No antagonistic reactions were observed between any two strains, and significant metabolic synergistic effects were observed. Cross-feeding validation results showed that Bacillus licheniformis MY176 had an OD value of [missing information - likely a value] in the supernatant of Lactobacillus reuteri MRY6. 600 The peak value was increased by 18.3%, and the logarithmic phase of Bacillus subtilis YCNL1 was prolonged by approximately 4 hours in the supernatant of Bacillus licheniformis MY176. Synergistic validation experiments conducted using the "one-out method" confirmed that the absence of any strain led to a decrease in the community's growth rate, a prolonged lag phase, or accelerated death. Key growth indicators such as maximum specific growth rate, comprehensive growth index, and total biomass showed varying degrees of decline, fully demonstrating the close synergy and indispensability of the four strains. The four strains have clearly defined functional divisions: Bacillus licheniformis MY176 consumes oxygen and produces cellulase and protease to degrade macromolecular substrates; Bacillus subtilis YCNL1 assimilates ammonia nitrogen to synthesize bacterial proteins; Lactobacillus reuteri MRY6 produces lactic acid to regulate pH and mediate immune regulation; and Enterococcus faecalis G1 produces acid and secretes enterococci to inhibit pathogenic bacteria. The functions of each strain are complementary and synergistic, jointly ensuring the overall effectiveness of the synthetic community. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The results of a pairwise plate antagonism experiment of four single bacteria are shown in the figure. Figure 2 The growth curves are shown for cross-feeding cross-validation of the interaction between a single bacterium and three other bacterial cultures. Figure A shows the growth curves of Bacillus subtilis YCNL1 single strain and the other three strains with Bacillus subtilis supernatant; Figure B shows the growth curves of Bacillus licheniformis MY176 single strain and the other three strains with Bacillus licheniformis supernatant; Figure C shows the growth curves of Lactobacillus reuteri MRY6 single strain and the other three strains with Lactobacillus reuteri supernatant; and Figure D shows the growth curves of Enterococcus faecalis G1 single strain and the other three strains with Enterococcus faecalis supernatant. Figure 3 The growth curves of each group in MRS medium; Figure 4 Bar chart showing the effect of single-strain deletion on microbial community growth indicators; Figure 5 Figure showing the effect of SynComs-R treatment on rumen microbial alpha diversity in periparturient dairy cows; Figure 6 Dilution curves showing the effect of SynComs-R treatment on total rumen microbiota in periparturient dairy cows; Figure 7 The left and right plots show the effects of SynComs-R treatment on rumen microbial β-diversity in peripartum dairy cows and their similarity analysis. The left plot is the principal coordinate analysis based on Bray-Curtis distance, and the right plot is the genus-level Anosim similarity analysis. Figure 8 Heatmap of SynComs-R cluster analysis of relative abundance of rumen microorganisms in dairy cows during the peripartum period; Figure 9 The left and right images show the abundance of rumen microbiota. The left image shows the abundance at the phylum level, and the right image shows the abundance at the species level. Figure 10 PCA analysis of the effect of SynComs-R on rumen metabolome levels in periparturient dairy cows; Figure 11 A diagram showing the predictive analysis of rumen KEGG pathway function; Figure 12 Figure showing the effect of SynComs-R on the alpha diversity of gut bacteria in peripartum dairy cows; Figure 13 Figure showing the effect of SynComs-R on rumen microbial alpha diversity in meat sheep; Figure 14 The diagram shows the abundance of rumen microbiota in sheep; the left diagram shows the abundance at the phylum level, and the right diagram shows the abundance at the genus level. Figure 15 Functional prediction diagram of KEGG_3 rumen microbiota in meat sheep; Figure 16 Figure showing the effect of SynComs-R on the alpha diversity of gut microbiota in meat sheep; Figure 17 The diagram shows the abundance of gut microbiota in sheep; the left diagram shows the abundance at the phylum level, and the right diagram shows the abundance at the genus level. Figure 18 Functional prediction diagram of KEGG_3 gut microbiota in meat sheep; Figure 19 This is a summary chart of the regulatory effects of SynComs-R on ruminant production performance, digestive metabolism, and health indicators. Detailed Implementation

[0024] The core strains involved in the following embodiments of the present invention were all isolated from rumen fluid and fecal samples of healthy ruminants and deposited at the China General Microbiological Culture Collection Center (CGMCC) and the China Center for Type Culture Collection (CCTCC), respectively. Specific information is as follows: Bacillus licheniformis MY176 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 11, 2026, with accession number CCTCCNO: M 2026425. Bacillus subtilis YCNL1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is November 17, 2023, and the accession number is CGMCC NO: 29050. Limosilactobacillus reuteri MRY6 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on September 23, 2024, with accession number CCTCC NO: M 20242030. Enterococcus faecalis G1 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 11, 2026, with accession number CCTCC NO: M2026424.

[0025] General Materials and Methods 1. Culture medium preparation Selection medium (LBS): 10g peptone, 10g beef extract, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 20g glucose, 5g calcium carbonate, 1ml Tween-80, 25g agar, 1000mL distilled water, pH 6.0-6.5, autoclaved at 121℃ for 30 minutes.

[0026] Isolation medium (MRS): 10g peptone, 8g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g disodium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 20g calcium carbonate, 14g agar, 1ml Tween-80, 1000mL distilled water, pH 6.5±0.2, sterilized at 121℃ for 30min.

[0027] 2. General Determination Method Growth curve determination: After activation, the bacterial culture was inoculated into the corresponding liquid culture medium and cultured in a growth curve analyzer. The absorbance (OD) of the strain was measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours. 600 .

[0028] Rumen fluid collection and processing: Rumen fluid was collected before morning feeding. The ruminant was secured with a neck clamp, and the rumen tube was inserted into the rumen along the corner of the mouth. The rumen fluid was drawn out with a syringe. The first tube, which was mainly composed of saliva, was discarded. The rumen fluid was then collected and the gastric tube was removed. The rumen fluid was filtered through four layers of gauze and then used for later use.

[0029] Growth performance determination: Record animal weight, milk production, feed intake and other indicators at the time points of the experimental design, and calculate parameters such as average daily weight gain, feed conversion ratio and average daily milk production.

[0030] Immunological markers were measured: inflammatory factors (IL-1β, IL-6, IL-10, TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA). The ELISA kit was purchased from Huayuechang Biotechnology Co., Ltd.

[0031] Gastrointestinal microbiota determination: Rumen fluid and fecal samples were sent to Beijing Novogene Technology Co., Ltd. for 16S rRNA gene sequencing using the Illumina NovaSeq 6000 high-throughput sequencing platform. Genomic DNA was extracted from the samples using a soil genomic DNA extraction kit (catalog number: DP336), and the concentration and purity of the genomic DNA samples were determined using a NanoDrop 2000 spectrophotometer. The V3-V4 region of 16S rDNA was amplified by polymerase chain reaction (PCR). Library construction was performed using The NEBNext Ultra II DNA Library Prep Kit (Cat.No.E7645B). High-quality sequence clustering of OTUs was performed using Vsearch software with a sequence similarity threshold of 97%. Representative sequences were compared with the Silva138 database to obtain species classification information for each OTU. α and β diversity analysis was performed using QIIME2 software.

[0032] Metabolomics analysis of digestive tract samples: Rumen fluid and fecal samples were collected under standardized conditions (rumen fluid was filtered, and fecal samples were mixed and aliquoted), flash-frozen in liquid nitrogen, and stored at -80℃ before being sent to the State Key Laboratory of Hebei Agricultural University for analysis. A high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) system was used as the core analytical platform. Separation was performed using an ACQUITY UPLCHSS T3 column, and data were acquired in the m / z range of 50-1000 using electrospray ionization in both positive and negative ion modes. Quality control samples were inserted for every 10 samples, leucine-endorphin quality correction, and daily instrument calibration were performed. The peak retention time RSD was <0.5%, and the peak intensity RSD for QC samples was <15%. Non-targeted identification of metabolites was performed using the HMDB and KEGG databases.

[0033] In vitro rumen fermentation parameter determination: Rumen buffer was prepared according to the method of Menke et al.; Gas production measurement: Gas was extracted using a syringe and the gas production was recorded at 2, 4, 6, 8, 10, 12, 24, 36, and 48 hours of in vitro fermentation. The gas production was then substituted into the gas production model GP = a + b(1 - e^(-a / b)). -ct Calculate the gas production parameters. Where t represents fermentation time (h), GP represents gas production at time t (mL), a represents gas production of the rapid degradation portion (mL), b represents gas production of the slow degradation portion (mL), c represents gas production rate of b (% / h), and a+b represents the theoretical total gas production (mL). Determination of nutrient degradation rate: The residue after fermentation for 48 hours was filtered through a 300-mesh nylon bag and dried at 65℃ for 48 hours. The dry matter (DM) and other indicators in the diet and fermentation residue were determined according to the AOAC (2000) method. Fermentation parameters were determined as follows: 5 mL of fermentation broth was taken into centrifuge tubes at 4, 8, 12, 24, 36, and 48 h of in vitro fermentation. The concentration of cell protein (MCP) was determined by the Coomassie brilliant blue method, and the concentration of ammonia nitrogen (NH3-N) was determined by the indophenol colorimetric method as described by Chaney et al.

[0034] Example 1: Screening and compatibility verification of strains within SynComs-R This embodiment is based on the rumen microbial strain library previously constructed by the research group (503 strains were isolated from rumen fluid and fecal samples of healthy ruminants, all of which have been identified). Probiotics included in the feed additive catalog were screened from the library, and systematic compatibility screening and verification were carried out.

[0035] 1.1 Initial screening: Functional classification of candidate strains From 503 bacterial strains, 172 probiotic strains listed in the feed additive catalog were screened. These strains all possess one or more core functions, including aerobic consumption, acid production, anti-inflammation, and enzyme production. They are specifically classified as follows: 47 oxygen-consuming strains: mainly Bacillus species, which can create a suitable anaerobic environment for strict anaerobic bacteria by consuming oxygen in the rumen; 53 acid-producing strains: mainly Lactobacillus, which can produce organic acids such as lactic acid and acetic acid through carbohydrate fermentation, thereby reducing the local pH value and inhibiting pathogens; Thirty-eight anti-inflammatory strains were identified, mainly from the genera *Lactobacillus* and *Bifidobacterium*. These strains could induce host cells to secrete IL-10 or inhibit the growth of pathogens by secreting bacteriocins and organic acids. Thirty-four enzyme-producing strains were identified, mainly belonging to the genus Bacillus, which can secrete extracellular enzymes such as cellulase, hemicellulase, protease, amylase, and pectinase to degrade complex polysaccharides and proteins.

[0036] 1.2 Compatibility Screening: Antagonism Test and Cross-feeding Test The 172 probiotic strains were paired up to form 14,706 groups, and antagonistic and cross-feeding experiments were carried out systematically.

[0037] The antagonism test results showed that among the 14,706 combinations, 14,288 combinations showed varying degrees of antagonism or growth competition (mainly manifested as competition for bacteriocins or nutrient metabolism) and were excluded; 418 combinations without antagonistic reactions were retained, involving 30 strains of bacteria.

[0038] A cross-feed experiment was conducted on the aforementioned 30 bacterial strains: the culture supernatant of one strain was extracted and added to the culture medium of another strain, and the growth curves were measured to screen for combinations exhibiting unidirectional or bidirectional growth-promoting effects, i.e., the metabolites in the supernatant could be utilized by the other strain or could create a favorable growth environment for the other strain. Ultimately, combinations exhibiting significant synergistic effects were screened, resulting in 18 candidate strains.

[0039] 1.3 Secondary screening: Multiple rounds of validation via in vitro rumen fermentation To verify the actual function of 18 candidate strains in the digestive tract environment of ruminants, multiple rounds of screening were conducted using an in vitro rumen fermentation system, gradually eliminating combinations with functional redundancy or poor environmental adaptability: In the first round of gas production measurement, 14 combinations with a cumulative gas production of ≥85mL / g over 48 hours and a theoretical maximum gas production of ≥95mL / g were retained. The second round of fermentation parameter determination retained five combinations with stable pH at 6.4-6.8, NH3-N concentration ≥35mg / dL, and MCP yield ≥3.3mg / mL; The third round of measurements determined the degradation rate of each fiber combination and comprehensively evaluated dynamic indicators such as oxygen consumption rate, acid production rate, and pH stability.

[0040] Four target strains were ultimately identified: Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1. The functional divisions of the four strains are as follows: Bacillus licheniformis MY176 consumes oxygen and produces cellulase and protease to degrade macromolecular substrates; Bacillus subtilis YCNL1 assimilates ammonia nitrogen to synthesize bacterial proteins and simultaneously produces acid to inhibit bacteria; Lactobacillus reuteri MRY6 produces lactic acid to lower pH and regulates host immunity through its metabolites; and Enterococcus faecalis G1 produces acid and secretes enterococcal toxins to inhibit pathogenic bacteria.

[0041] 1.4 Compatibility Verification (1) Plate antagonism test like Figure 1 As shown, no growth inhibition bands appeared in the streak plate culture results of the four single bacteria in pairs, proving that there was no obvious antagonistic reaction among the four strains, which meets the basic premise of compound compatibility.

[0042] (2) Cross-feed cross-validation experiment like Figure 2 As shown, the logarithmic growth phase OD values ​​of each strain after the addition of supernatant from other strains were observed. 600 Both peak and plateau cell densities were higher than those of the single-strain control; among them, Bacillus licheniformis MY176 had a higher OD in the supernatant of Lactobacillus reuteri MRY6. 600 The peak value was increased by 18.3%, and the logarithmic phase of Bacillus subtilis YCNL1 was prolonged by about 4 hours in the supernatant of Bacillus licheniformis MY176, confirming a significant synergistic growth effect among the four strains. Based on this, the synthetic microbial community SynComs-R was constructed.

[0043] In summary, this embodiment obtained four target strains with synergistic and complementary functions from 172 candidate bacteria with core functions through 14,706 combinations of antagonistic tests and multiple rounds of in vitro screening. The verification showed that the four strains had no antagonistic reaction and had a significant metabolic synergistic effect, providing a scientific basis for the construction of SynComs-R.

[0044] Example 2: Verification of the synergistic effect of SynComs-R four bacteria This embodiment uses the "removal of one strain" method to verify the synergistic effect of the four strains and clarify the overall growth performance advantage of SynComs-R.

[0045] 2.1 Experimental Design The test strains were Bacillus licheniformis MY176 (denoted as A), Bacillus subtilis YCNL1 (denoted as B), Lactobacillus reuteri MRY6 (denoted as C), and Enterococcus faecalis G1 (denoted as D). All strains were activated and co-cultured using MRS liquid medium.

[0046] The experiment consisted of 6 treatment groups, with 6 replicates per group, and the total inoculation amount for each group was controlled at 1×10⁻⁶. 9 CFU / tube to ensure consistent initial total biomass; specific grouping and inoculation protocols are shown in Table 1.

[0047] Table 1 Trial Groups and Vaccination Protocols

[0048] Note: A is Bacillus licheniformis MY176, B is Bacillus subtilis YCNL1, C is Lactobacillus reuteri MRY6, and D is Enterococcus faecalis G1.

[0049] 2.2 Measurement and Analysis Methods The inoculated test tubes were placed in a fully automated microbial growth curve analyzer, with the temperature set at 37℃ and the rotation speed at 180 rpm. The absorbance value (OD) at a wavelength of 600 nm was automatically measured and recorded every 1 hour. 600 ), continuously monitored for 48 hours.

[0050] The growth curves of each group were fitted using the Gompertz model, and three key growth parameters were extracted: lag phase (λ, reflecting the speed at which the bacterial community adapts to the environment), maximum specific growth rate (μmax, reflecting the growth and reproduction rate of the bacterial community), and maximum biomass (A, reflecting the strongest growth density of the bacterial community). The comprehensive growth performance index 1 / Tmid was calculated using the above parameters. The larger the value, the stronger the growth ability of the strain.

[0051] 2.3 Test Results like Figure 3 As shown, the four-strain co-culture group (ABCD) maintained high biomass and balanced growth rate during both the logarithmic growth phase and the stationary phase; however, when any strain was missing, each group exhibited varying degrees of performance defects, such as decreased growth rate, prolonged lag phase, or accelerated death, indicating that the four-strain combination has the optimal synergistic growth effect.

[0052] like Figure 4As shown, the four-strain co-culture group (ABCD) had a baseline value of 1.0 for the maximum specific growth rate (μmax), the comprehensive growth index (1 / Tmid), and the total biomass (AUC). The absence of any one strain would lead to a decrease in at least one key indicator. Specifically, the absence of Enterococcus faecalis G1 (D) and Lactobacillus reuteri MRY6 (C) significantly reduced μmax and 1 / Tmid, demonstrating that the four-strain combination has the best synergistic growth performance and that none of them can be omitted.

[0053] This embodiment uses the "one-out method" design verification. The results show that the absence of any strain leads to a significant decrease in key indicators of microbial community growth, indicating that there is a close synergistic effect among the four strains. SynComs-R is a functionally synergistic and indispensable whole of microbial functions, rather than a simple superposition of the functions of individual strains.

[0054] Example 3: Comparative experiment of in vitro rumen fermentation between SynComs-R group and single-strain group This embodiment compares the regulatory effects of SynComs-R and various single strains on rumen fermentation through in vitro rumen fermentation experiments, verifying the synergistic effect. The experiment was conducted in the rumen microbial culture bank laboratory of Hebei Agricultural University from May to June 2025.

[0055] 3.1 Experimental Materials and Design Frozen *Lactobacillus reuteri* MRY6, *Bacillus subtilis* YCNL1, *Bacillus licheniformis* MY176, and *Enterococcus faecalis* G1 were activated. The experiment included one control group (CON, no strain added), four single-strain addition groups (*Bacillus licheniformis* group, *Bacillus subtilis* group, *Lactobacillus reuteri* group, and *Enterococcus faecalis* group), and one SynComs-R group, with six replicates in each group.

[0056] Fresh rumen fluid was collected from the designated sheep slaughterhouse of Guofu Tangyao Meat Products Co., Ltd. in Tang County, Baoding, Hebei Province. After being filtered through four layers of gauze, it was mixed evenly with rumen buffer at a ratio of 1:2 and injected into 500mL fermentation bags. 6g of basal feed was used as the fermentation substrate. The fermentation bags were deoxygenated and sealed using a vacuum packaging machine. The fermentation bags were then placed in a 39℃ constant temperature water bath for in vitro rumen fermentation at a speed of 45r / min for 48h.

[0057] 3.2 Test Results (1) Impact on gas production and gas production parameters The effects of SynComs-R and individual strains on in vitro fermentation gas production are shown in Table 2.

[0058] Table 2. Effects of SynComs-R single strains on gas production during in vitro fermentation.

[0059] Note: Different lowercase letters in the same row of data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05); a represents the gas production of the rapid degradation portion, b represents the gas production of the slow degradation portion, c represents the gas production rate of b, and a+b represents the theoretical maximum gas production; "-" indicates gas production lag, and the gas production of a is expressed in absolute value.

[0060] As shown in Table 2, the gas production and theoretical maximum gas production of the SynComs-R group at 48h were significantly higher than those of the control group (P<0.01) and each single strain group (P<0.05), respectively, increasing by 15.6% and 10.4% compared with the control group, indicating that the synergistic fermentation effect of SynComs-R is significantly better than that of a single strain.

[0061] (2) Effects on rumen fermentation parameters The effects of SynComs-R and individual strains on rumen fermentation parameters are shown in Table 3.

[0062] Table 3. Effects of SynComs-R single strains on rumen fermentation parameters

[0063] Note: Different lowercase letters in the superscript of data in the same row indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05); NH3-N is the concentration of ammonia nitrogen, and MCP is the yield of bacterial protein.

[0064] As shown in Table 3, while maintaining stable rumen pH, the SynComs-R group showed a 7.03% increase in ammonia nitrogen (NH3-N) concentration and a 10.34% increase in microbial protein (MCP) production compared to the control group (P<0.05).

[0065] (3) Effect on nutrient degradation rate The effects of SynComs-R and individual strains on nutrient degradation rates are shown in Table 4.

[0066] Table 4. Effects of SynComs-R comparative single strains on nutrient degradation rate

[0067] Note: Different lowercase letters in the same row of data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05); DM is dry matter, EE is crude fat, CP is crude protein, NDF is neutral detergent fiber, and ADF is acid detergent fiber.

[0068] As shown in Table 4, compared with the control group, the degradation rate of neutral detergent fiber NDF in the SynComs-R group increased by 3.29%, and the degradation rate of crude fat EE increased by 2% (P<0.05).

[0069] In summary, the in vitro fermentation experiment showed that, compared with each single-strain group, the SynComs-R group exhibited superior performance in terms of gas production, cell protein synthesis, and fiber degradation. The synergistic effect of the four strains in equal proportions was significantly better than that of any single strain, verifying the scientific compatibility and functional synergistic advantages of the present invention.

[0070] Example 4: Application of SynComs-R in peripartum dairy cow feeding This study used peripartum Holstein dairy cows as experimental animals and a microbiota transplantation group as a positive control to verify the regulatory effects of SynComs-R on peripartum dairy cow production performance, rumen fermentation, intestinal health, and immune status. Microbiota transplantation is currently recognized as a highly effective technique for regulating the digestive health and improving growth performance of ruminants. Using this as a control clearly demonstrates the advantages of SynComs-R. The experiment was conducted from November 2024 to January 2025 at Junlebao Jiasi Ranch, Weixian County, Xingtai City, Hebei Province.

[0071] 4.1 Experimental Design Forty-five Holstein dairy cows with good health, identical parity (second-parity cows), and similar weight, body condition, and expected calving date were selected and randomly divided into three groups of 15 cows each: Control group (CON): Dairy cows were given 10L of warm water immediately after calving; SynComs-R group: Immediately after calving, dairy cows were administered 9.8L of physiological saline and 0.2L of SynComs-R mixed bacterial solution (containing Bacillus subtilis YCNL1, Bacillus licheniformis MY176, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1, four strains mixed in equal amounts). Positive control group (RMT): dairy cows were given 10L of in vitro expanded rumen fluid immediately after calving.

[0072] Three healthy dairy cows at peak lactation with a daily milk yield of 45 kg were selected as rumen fluid donors (DCG / DON). 500 mL of rumen fluid was collected from each cow and placed in a thermos. 2 mL of the rumen fluid from each cow was stored at -80℃ for 16S rDNA assay. The overall experimental period was 28 days postpartum.

[0073] After calving, the experimental cows received timely veterinary care and were transferred to the postpartum pen. The cows had free access to feed and water; the diet composition and nutrient levels are shown in Table 5. The experimental cows were fed twice daily (08:00 and 16:00), and milking began on the third day after calving, three times daily (06:00, 14:00, and 20:00). The cows were housed in a dry, well-ventilated, and comfortable environment, which was regularly cleaned and disinfected during the experiment.

[0074] Table 5. Composition of raw materials and basic nutrients in the experimental diets (dry matter basis)

[0075] Note: ① The premix provides 30,000 IU of vitamin A, 7,500 IU of vitamin D, 1,200 IU of vitamin E, 45 mg of Cu, 150 mg of Zn, 300 mg of Fe, 150 mg of Mn, 0.9 mg of Se, 3 mg of I, and 0.6 mg of Co per kilogram of diet; ② Net energy for lactation is a calculated value, and all other nutrients are measured values.

[0076] 4.2 Test Results (1) Impact on milk production The effects of SynComs-R on milk production in periparturient dairy cows 108 days postpartum are shown in Table 6.

[0077] Table 6. Effects of SynComs-R on milk yield in dairy cows 108 days postpartum (kg / d)

[0078] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0079] As shown in Table 6, the milk production and average daily milk production of the SynComs-R group from 3 to 108 days postpartum were significantly higher than those of the CON group (P < 0.05), and the average daily milk production was 10% higher than that of the CON group (4.11 kg / d), while also being significantly better than that of the RMT group with microbial transplantation.

[0080] (2) Effects on apparent digestibility of nutrients The effect of SynComs-R on the apparent digestibility of nutrients in peripartum dairy cows is shown in Table 7.

[0081] Table 7. Effects of SynComs-R on the apparent digestibility of nutrients in peripartum dairy cows

[0082] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0083] As shown in Table 7, the apparent digestibility of crude protein (CP) in the SynComs-R group was 7.11% higher than that in the control group (CON group), and the apparent digestibility of neutral detergent fiber (NDF) was 10.86% higher (P < 0.05); compared with the RMT group, the apparent digestibility of CP was 3.04% higher.

[0084] (3) Effects on rumen juice fermentation parameters The effects of SynComs-R on rumen fluid fermentation parameters in peripartum dairy cows are shown in Table 8.

[0085] Table 8. Effects of SynComs-R on rumen fermentation parameters in periparturient dairy cows

[0086] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0087] As shown in Table 8, the MCP content in the SynComs-R group was 2.65% higher than that in the control group (P < 0.05), and the rumen pH value remained stable (P > 0.05).

[0088] (4) Effects on blood inflammatory factors The effects of SynComs-R on blood inflammatory factors in peripartum dairy cows are shown in Table 9.

[0089] Table 9. Effects of SynComs-R on blood inflammatory factors in peripartum dairy cows.

[0090] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0091] As shown in Table 9, compared with the microbiome transplantation RMT group, the SynComs-R group showed a 7.08% decrease in the pro-inflammatory factor TNF-α and an 8.83% increase in the anti-inflammatory factor IL-10 (P<0.05), indicating that SynComs-R can effectively alleviate the systemic inflammatory response in peripartum dairy cows and has a better overall anti-inflammatory effect than the microbiome transplantation group.

[0092] (5) Effects on volatile fatty acids (VFA) in rumen fluid The effects of SynComs-R on VFA in rumen fluid of peripartum dairy cows are shown in Table 10.

[0093] Table 10. Effects of SynComs-R on VFA in Rumen Fluid of Dairy Cows During the Perifum Period

[0094] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0095] As shown in Table 10, the total volatile acids in the SynComs-R group increased by 12.77% compared to the control group, butyric acid increased by 49.77%, and propionic acid increased by 20.14% compared to the microbial transplantation RMT group, indicating a better composition of volatile fatty acids.

[0096] (6) Effects on the rumen microbial community like Figure 5 As shown, the Chao1 richness, Shannon diversity, and Evenness evenness of the rumen microbiota in the SynComs-R group were increased by 12.5%, 18.8%, and 18.9% respectively compared with the control group (CON) (P<0.001), and all were higher than those of the donor cow group at the peak of lactation (DCG). This indicates that SynComs-R can promote the rapid development of rumen microbiota in peripartum dairy cows and reach the microbiota structure level at the peak of lactation.

[0097] like Figure 6 The dilution curve results showed that the number of rumen microbial OTUs in the SynComs-R group was about 17.7% higher than that in the CON group, and exceeded the level in the DCG group at the peak of lactation.

[0098] like Figure 7 As shown, the sample points from the donor cow group (DCG) and the SynComs-R group at peak lactation were significantly different from those from the CON group, and all sample points from the DCG group were located within the SynComs-R group. Genus-level Anosim similarity analysis further confirmed that the rumen microbiota structure of the SynComs-R group and the DCG group was at the same level of similarity, indicating that SynComs-R can rapidly bring the rumen microbiota of peripartum dairy cows to a stable structure at peak lactation.

[0099] like Figure 8 As shown, the SynComs-R group samples clustered together with the DCG group at the peak of lactation, and the relative abundance distribution of the main dominant bacterial genera (such as Prevotella) was consistent with that of the DCG group, further indicating that SynComs-R can enable the peripartum rumen microbiota of dairy cows to rapidly develop to the structural characteristics of the peak lactation period.

[0100] like Figure 9 As shown, at the phylum level, the abundance of Bacteroidota and Bacillota in the SynComs-R group of rumen microbes was significantly higher than that in the peak lactation donor group (DCG) and the control group (CON); at the species level, the abundance of species related to protein metabolism (such as Prevotella) increased, indicating that SynComs-R can optimize the composition of rumen microbes and improve feed digestibility and metabolic health in peripartum dairy cows.

[0101] (7) Effects on the rumen metabolome like Figure 10 As shown, the control group (CON) and the SynComs-R group were completely separated on the PC1 axis, and the samples within each group were closely clustered with high intragroup consistency, indicating that SynComs-R treatment significantly altered the rumen metabolic characteristics of peripartum dairy cows and triggered metabolic remodeling.

[0102] like Figure 11 As shown, compared with the control group (CON), SynComs-R significantly upregulated the rumen glycolysis / gluconeogenesis and pyruvate metabolism pathways (P<0.05). In the volatile fatty acid (VFA) generation pathway, SynComs-R promoted the conversion of pyruvate to oxaloacetate and enhanced the succinate pathway, thereby significantly increasing the propionic acid ratio. Simultaneously, by activating the butyrate kinase pathway, it promoted the conversion of acetyl-CoA to butyrate. This molecularly explains the mechanism behind the significantly increased propionic and butyrate ratios in the SynComs-R group, providing the host with a more efficient energy substrate.

[0103] (8) Effects on volatile fatty acids in the intestine The effect of SynComs-R on the intestinal VFA concentration of peripartum dairy cows is shown in Table 11.

[0104] Table 11 Effects of SynComs-R on VFA concentration in the intestines of dairy cows during the peripartum period

[0105] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0106] As shown in Table 11, the total volatile acids in the SynComs-R group increased by 3.99% and butyrate by 6.9% compared to the control group (CON). Butyrate, as a major energy substrate that can be directly utilized by intestinal epithelial cells, can preferentially supply energy to the colonic epithelium and maintain intestinal barrier function, indicating that SynComs-R can improve the intestinal energy metabolism status of peripartum dairy cows.

[0107] (9) Effects on the gut microbiota The effects of SynComs-R on the phylum-level gut bacterial composition of peripartum dairy cows are shown in Table 12.

[0108] Table 12 Effects of SynComs-R on the phylum-level gut bacterial composition of periparturient dairy cows

[0109] Note: Different lowercase letters on the shoulder labels of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05); DON refers to donor cows at peak lactation.

[0110] As shown in Table 12, the relative abundance of Firmicutes, the dominant bacterial group in the SynComs-R group, was 10.82% higher than that in the control group (CON), and was close to the DON level during peak lactation (P<0.001). This indicates that SynComs-R can effectively promote the recovery of the gut microbiota of dairy cows during the peripartum period to the structure during peak lactation, improve the balance of the microbiota, and thus improve production performance.

[0111] The effects of SynComs-R on the genus-level gut bacterial composition of peripartum dairy cows are shown in Table 13.

[0112] Table 13 Effects of SynComs-R on the genus-level gut bacterial composition of peripartum dairy cows

[0113] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0114] Table 13 shows that SynComs-R significantly altered the gut microbiota structure: the abundance of butyrate-producing bacteria genus UCG-005 increased by 28.97% compared to the control group (CON), and the abundance of Agathobacter increased by 85.06%, approaching the level of donor cows at peak lactation (DON); while the abundance of the potentially pathogenic Escherichia-Shigella complex decreased by 97.89% compared to the control group (P<0.001). This indicates that SynComs-R can improve animal production performance and health by enriching butyrate-producing bacteria and inhibiting pathogenic bacteria, while simultaneously promoting the restoration of beneficial gut microbiota in peripartum dairy cows to the structure of peak lactation.

[0115] like Figure 12 As shown, compared with the control group (CON), the SynComs-R group showed significantly improved richness and diversity of gut microbiota (Shannon and Evenness), approaching the level of the DON group at the peak of lactation. This indicates that SynComs-R treatment can effectively promote the rapid development of gut microbiota in dairy cows during the peripartum period and restore the microbiota structure characteristics to the peak of lactation.

[0116] Example 5: Application of synthetic microbial communities in sheep farming This embodiment uses Duhu sheep as experimental animals to verify the regulatory effect of SynComs-R on the growth performance, digestive metabolism, rumen and intestinal flora and inflammatory status of meat sheep.

[0117] 5.1 Experimental Design Sixty-four 2-month-old Duhu sheep with similar body weights (16.0±1.8 kg) were randomly divided into a CON group and a SynComs-R group, with 32 replicates in each group. The CON group was fed a basal diet, while the SynComs-R group was fed a basal diet supplemented with ≥1.0×10 10 CFU / kg SynComs-R bacterial solution prepared in this invention (containing Bacillus subtilis YCNL1, Bacillus licheniformis MY176, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1, four strains mixed in equal amounts).

[0118] The trial lasted for 60 days, including a 14-day pre-trial period (during which 7.5% of the original diet was replaced daily in the experimental diet) and a 56-day formal trial period. The concentrate-to-roughage ratio of the diet was 7:3 from 0 to 28 days, and the composition and nutritional levels are shown in Table 14. The concentrate-to-roughage ratio of the diet was 9:1 from 28 to 56 days, and the composition and nutritional levels are shown in Table 15.

[0119] Before each feeding, the bacterial solution was thoroughly mixed with the feed to ensure that all the bacterial solution was consumed. The diets for all experimental sheep were isoenergetic and isonitrogenous, with consistent nutritional levels. The sheepfold and feeding passage were thoroughly disinfected before the sheep entered the pen. After entering the pen, the sheep were tagged with ear tags. They were fed twice daily, at 7:00 AM and 5:00 PM, with free access to feed and water. The amount of feed consumed and any uneaten feed were recorded daily, along with the sheep's mental state and fecal condition.

[0120] On the last day of the experiment, before morning feeding, 20 mL of rumen fluid was collected from each sheep, filtered through four layers of gauze, and the pH was measured. The fluid was then stored at -20°C for rumen fermentation and microbial parameter determination. Blood was collected from the jugular vein of 15 sheep in each group (5 mL / sheep), centrifuged at 3000 rpm for 10 min, and the supernatant was stored at -20°C for serum biochemical assays. Feces were collected every 6 hours for the last 3 days of the experiment, and after nitrogen fixation, were used for digestibility and rectal microbial assays. During the experiment, feed was collected from each group weekly and stored at -20°C for nutrient composition analysis.

[0121] Table 14 Composition and nutrient levels of a 7:3 concentrate-to-roughage ratio basal diet (dry matter basis)

[0122] Note: (1) The premix is ​​provided per kilogram of feed: Vitamin A 2400 IU, Vitamin D 620 IU, Vitamin E 40 mg, Copper 8.5 mg, Zinc 55 mg, Iron 35 mg, Manganese 52 mg, Selenium 0.35 mg, Iodine 0.5 mg, Cobalt 0.3 mg; (2) Metabolizable energy is calculated, and all others are measured values.

[0123] Table 15 Composition and nutrient levels of basal diets with a concentrate-to-roughage ratio of 9:1 (dry matter basis)

[0124] Note: (1) The premix is ​​provided per kilogram of feed: Vitamin A 2400 IU, Vitamin D 620 IU, Vitamin E 40 mg, Copper 8.5 mg, Zinc 55 mg, Iron 35 mg, Manganese 52 mg, Selenium 0.35 mg, Iodine 0.5 mg, Cobalt 0.3 mg; (2) Metabolizable energy is calculated, and all others are measured values.

[0125] 5.2 Test Results (1) Effects on growth performance The effects of SynComs-R on the growth performance of meat sheep are shown in Table 16.

[0126] Table 16 Effects of SynComs-R on the growth performance of meat sheep

[0127] As shown in Table 16, compared with the control group, the SynComs-R group showed an average daily weight gain of 14.8% at the end of 0-28 days (P<0.05) and an average daily weight gain of 20.5% at 28-56 days (P<0.01); at the same time, the feed conversion ratio decreased by 6.3% and 9.0%, respectively, indicating that SynComs-R can significantly promote the growth performance of meat sheep and improve feed conversion efficiency.

[0128] (2) Effect on nutrient digestibility The effects of SynComs-R on the digestibility of sheep are shown in Table 17.

[0129] Table 17 Effects of SynComs-R on digestibility indicators of mutton sheep

[0130] As shown in Table 17, SynComs-R significantly improved the nutrient digestibility of sheep, with dry matter (DM) digestibility increasing by 7.4% and CP digestibility increasing by 6.8% (P<0.01).

[0131] (3) Effects on rumen fermentation indicators The effects of SynComs-R on rumen fermentation parameters in sheep are shown in Table 18.

[0132] Table 18 Effects of SynComs-R on Rumen Fermentation Indicators in Meat Sheep

[0133] As shown in Table 18, SynComs-R significantly increased the synthesis of microbial protein (MCP) in meat sheep, by 13.82% compared with the control group (P<0.01).

[0134] (4) Effects on serum inflammatory factors The effects of SynComs-R on serum inflammatory factors in mutton sheep are shown in Table 19.

[0135] Table 19 Effects of SynComs-R on serum inflammatory factors in sheep.

[0136] As shown in Table 19, compared with the control group, the SynComs-R group showed a 56.5% decrease in IL-6, a 47.0% decrease in TNF-α, and a 50.9% decrease in IL-1β (P<0.01), demonstrating a significant anti-inflammatory effect.

[0137] (5) Effects on the rumen microbial community like Figure 13 As shown, the abundance of Chao1, the diversity of Shannon, and the evenness of rumen microorganisms in the SynComs-R experimental group were significantly lower than those in the control group (CON) (P<0.05), indicating that SynComs-R treatment improves rumen fermentation efficiency by reducing the redundancy and diversity of the microbial community and optimizing the composition of the core functional microbial community.

[0138] like Figure 14 As shown, at the phylum level, the relative abundance of Firmicutes in the experimental group was significantly higher than that in the control group, while the abundance of Bacteroidetes was correspondingly lower. At the genus level, the abundance of unclassified_Muribaculaceae was significantly increased (P<0.05), and the abundance of Prevotella showed an upward trend, indicating that SynComs-R treatment effectively remodeled the rumen microbial community structure and enhanced the fiber degradation function.

[0139] like Figure 15 As shown, compared with the control group, the SynComs-R group showed a significant increase in the relative abundance of key metabolic pathways such as anesamcinol biosynthesis, bacterial chemotaxis, pentose phosphate pathway, and glycerophospholipid metabolism (P<0.05), indicating that the addition of compound probiotics can enhance the metabolic activity and material synthesis capacity of rumen microorganisms, and promote energy metabolism and cell membrane construction.

[0140] (6) Effects on the gut microbiota like Figure 16 As shown, the abundance of Chao1, Shannon diversity, and evenness of gut microbiota in the SynComs-R experimental group were all higher than those in the control group, indicating that SynComs-R enriches the diversity of the hindgut microecology by introducing beneficial bacteria, thereby enhancing gut health and immune function.

[0141] like Figure 17As shown, the abundance of Bacillota in the gut microbiota of the experimental group was significantly higher than that of the control group (P<0.05); at the same time, the relative abundance of butyrate-producing beneficial bacteria genera such as UCG-005, Alistipes, and Christensenellaceae_R-7_group was significantly increased (P<0.05), indicating that the addition of compound probiotics can effectively promote the colonization of beneficial gut bacteria and optimize the microbial community structure.

[0142] like Figure 18 As shown, the core nutritional metabolic pathways such as butyrate metabolism, pyruvate metabolism, and tryptophan metabolism were significantly upregulated in the experimental group, while the lipopolysaccharide biosynthesis pathway was significantly downregulated. This can enhance intestinal nutritional metabolism, improve the digestibility of dry matter and crude protein, exert significant anti-inflammatory effects, reduce endotoxin production, lower serum pro-inflammatory factor levels, and improve the intestinal health of meat sheep.

[0143] Example 6: Application of synthetic microbial communities in beef cattle feeding This embodiment uses beef cattle as experimental animals to further verify the cross-species application effect of SynComs-R. The experiment was carried out at Yehui Livestock Co., Ltd. in Dingzhou City, Hebei Province from August to September 2025.

[0144] 6.1 Experimental Design 160 healthy beef cattle of similar weight and condition were selected and randomly divided into two groups of 80 each: Control group (CON): fed with basal diet; SynComs-R group: Add ≥1.0×10 to the basal diet 10 CFU / kg SynComs-R bacterial solution prepared in this invention (containing Bacillus subtilis YCNL1, Bacillus licheniformis MY176, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1, four strains mixed in equal amounts).

[0145] The experiment lasted for 30 days. During this period, the cattle had free access to feed and water and were kept in a dry, well-ventilated, and comfortable environment with regular cleaning and disinfection. Feed was collected from each group weekly and stored at -20℃ for nutrient analysis. Feces were collected every 6 hours for the last 3 days of the experiment, and after nitrogen fixation, they were used for digestibility testing. The body weight of the beef cattle at the beginning and end of the experiment was recorded, and growth performance indicators were calculated.

[0146] 6.2 Test Results (1) Effect on growth performance The effects of SynComs-R on the growth performance of beef cattle are shown in Table 20.

[0147] Table 20 Effects of SynComs-R on growth performance of beef cattle

[0148] As shown in Table 20, compared with the control group, the SynComs-R group had an average daily weight gain of 33.6% (P<0.01), demonstrating a significant growth-promoting effect.

[0149] (2) Effect on nutrient digestibility The effects of SynComs-R on the digestibility of beef cattle are shown in Table 21.

[0150] Table 21 Changes in SynComs-R indicators of beef cattle digestibility

[0151] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0152] As shown in Table 21, compared with the control group, the digestibility of DM in the SynComs-R group increased by 27.81%, the digestibility of EE increased by 5.05% (P<0.01), the apparent digestibility of NDF increased by 10.43%, and the apparent digestibility of ADF increased by 11.24% (P<0.05).

[0153] In summary, the beef cattle feeding trial showed that SynComs-R has significant growth-promoting and digestion-promoting effects. During the 30-day trial period, the average daily weight gain of beef cattle was 33.6% higher than that of the control group, and the digestibility of dry matter and fiber was significantly improved. Combined with the results of the peripartum dairy cattle and meat sheep trials, SynComs-R has a universal positive effect on ruminants across species and physiological stages.

[0154] Example 7: Comprehensive Analysis of SynComs-R Regulation Effect like Figure 19 As shown, in terms of digestibility, SynComs-R significantly improves the digestibility of crude protein, neutral detergent fiber, and dry matter, with a 27.81% increase in DM digestibility in beef cattle. Regarding growth performance, daily weight gain in sheep and beef cattle at different stages increased by 14.80%, 20.50%, and 33.60%, respectively. In terms of digestive health, serum pro-inflammatory factor TNF-α in sheep decreased by 47.0%, and IL-1β and IL-6 decreased by 50.90% and 56.50%, respectively. In terms of milk production, average daily milk yield in peripartum dairy cows increased by 9.97% from days 3 to 108. Regarding rumen metabolism, rumen butyrate levels in peripartum dairy cows increased by 49.77%. Overall, it exhibits positive regulatory effects across species and physiological states.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. 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 be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A synthetic microbial community SynComs-R for ruminants, characterized in that, The study consists of four functionally complementary strains: Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus reuteri MRY6, and Enterococcus faecalis G1. The preservation number of Bacillus licheniformis MY176 is CCTCC NO: M 2026425, the preservation number of Bacillus subtilis YCNL1 is CGMCC NO: 29050, the preservation number of Lactobacillus reuteri MRY6 is CCTCC NO: M 20242030, and the preservation number of Enterococcus faecalis G1 is CCTCC NO: M 2026424.

2. The SynComs-R synthetic microbial community according to claim 1, characterized in that, The viable count ratio of Bacillus licheniformis MY176, Bacillus subtilis YCNL1, Lactobacillus reuteri MRY6 and Enterococcus faecalis G1 was (0.5~1.5):(0.5~1.5):(0.5~1.5):(0.5~1.5).

3. A microbial inoculant, characterized in that, It includes the synthetic microbial community SynComs-R as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is in the form of a liquid agent, a freeze-dried powder, or microcapsule granules; the total viable count in the liquid agent is not less than 1 × 10⁻⁶. 8 CFU / mL.

5. A ruminant feed additive, characterized in that, It comprises the synthetic microbial community SynComs-R of claim 1 or the microbial agent of claim 3.

6. The ruminant feed additive according to claim 5, characterized in that, It also includes feed additives that are acceptable in terms of feed science.

7. The use of the SynComs-R synthetic microbial community as described in claim 1 in the preparation of products for optimizing rumen fermentation in ruminants and promoting gut health.

8. The application according to claim 7, characterized in that, The optimization of rumen fermentation and promotion of intestinal health in ruminants specifically includes: achieving bidirectional regulation across physiological states, correcting intestinal flora imbalance and restoring the flora to normal physiological levels under stress and abnormal conditions, and improving animal production performance and nutrient digestion efficiency under normal physiological conditions; achieving differentiated regulation at two sites in the rumen and gut, reducing rumen flora redundancy, selectively enriching fiber-degrading core flora to improve fermentation efficiency, increasing intestinal flora diversity, enriching butyrate-producing beneficial bacteria and inhibiting pathogenic bacteria to enhance the intestinal immune barrier; increasing fiber degradation rate and bacterial protein synthesis, selectively regulating volatile fatty acid composition to increase the proportion of propionic and butyrate, autonomously stabilizing rumen pH, inhibiting the proliferation of harmful intestinal bacteria, enhancing intestinal barrier function, and exerting systemic anti-inflammatory effects.

9. The application according to claim 7, characterized in that, The product is a feed additive for ruminants.

10. The application according to claim 7, characterized in that, The ruminants include dairy cows, sheep, and cattle.

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