Method for enriching fungi from gastrointestinal tract contents of ruminants and application of method

Abstract

The invention discloses a method for enriching fungi from gastrointestinal tract contents of ruminants and application of the method, and relates to the technical field of microbial separation. Comprising the following steps: screening and confirming a target sampling part, pre-treating and physically screening wrinkled stomach contents, carrying out compound enzyme digestion treatment, enriching fungal cells through sucrose density gradient centrifugation, deeply purifying the fungal cells and verifying an enrichment effect. The method comprises the following steps: firstly, determining that the rumphalia is a target part with the most abundant fungi through fungus specific qPCR analysis, then carrying out physical screening and vacuum filtration on the content of the rumphalia, then digesting by adopting a compound enzyme solution, dissociating the combination of the fungi and a plant matrix and degrading background DNA, and finally, carrying out quantitative analysis by utilizing a sucrose density gradient centrifugation technology to obtain the rumphalia. According to the method, fungal cells enriched in 30%, 45% and 60% concentration layers are selectively collected, and finally PCR and qPCR verification proves that the method can significantly improve the detection sensitivity and specificity of the fungal ITS gene.

Description

Technical Field

[0001] This invention relates to the field of microbial isolation technology, and in particular to a method for enriching fungi from the gastrointestinal contents of ruminants and its application. Background Technology

[0002] The gastrointestinal tract of ruminants (such as dairy cows) constitutes a complex and dynamic micro-ecosystem, home to a vast community of microorganisms including bacteria, archaea, protozoa, and fungi. These microorganisms form a close symbiotic relationship with the host, jointly participating in key physiological processes such as feed digestion, nutrient synthesis and absorption, energy metabolism, and immune regulation. Although advancements in molecular biology techniques have improved our understanding of bacterial and archaea communities, research on gastrointestinal fungi has long lagged behind. These fungi, especially anaerobic fungi (such as Neocallimastigomycota), are considered among the most efficient microbial groups in degrading plant lignocellulose. They produce powerful and diverse enzyme systems to break down complex plant cell wall polysaccharides, providing energy and substrates for both the host and the microbial community. Therefore, in-depth analysis of the community structure and function of gastrointestinal fungi and their correlation with host health and production performance is of significant scientific and applied value for optimizing ruminant nutrition, developing novel feed additives, and utilizing their enzyme systems for biomass conversion.

[0003] However, the primary technical bottleneck in accurately studying gastrointestinal fungi lies in their effective isolation and enrichment. Fungi have a much lower biomass in intestinal contents than bacteria, and their hyphae often tightly entwine and invade plant particles, forming physical bonds that make simple washing insufficient to release target cells. Furthermore, samples contain strong background interference from bacteria, host cells, feed residue, and cell-free DNA. The proportion of bacterial DNA in directly extracted total DNA is usually overwhelmingly dominant, severely interfering with the accuracy and sensitivity of subsequent fungi-specific molecular analyses (such as ITS region PCR, qPCR, and high-throughput sequencing).

[0004] In existing technologies, researchers have attempted various methods to address this problem. Direct culture methods are demanding and have limited representativeness; universal DNA extraction methods cannot increase the proportion of fungal DNA in total DNA; simple physical separation methods struggle to effectively distinguish fungi from impurities; and single-enzyme treatment methods cannot cope with the complexity of the gastrointestinal contents matrix. Therefore, there is an urgent need in the field for a pretreatment method that can efficiently and specifically enrich fungal cells while maximally removing background interference, laying the foundation for subsequent acquisition of high-purity fungal genomic DNA and reliable community and functional analysis. Therefore, this invention proposes a method for enriching fungi from the gastrointestinal contents of ruminants and its application to solve the problems existing in the prior art. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to propose a method for enriching fungi from the gastrointestinal contents of ruminants and its application. This method employs a combined strategy of physical sieving, complex enzymatic digestion, and density gradient centrifugation to selectively enrich target fungi while maximally removing bacteria, host cells, and physical impurities. This lays the foundation for obtaining high-purity fungal genomic DNA downstream, solving the problem in existing technologies where low fungal biomass, close binding to plant materials, and severe bacterial background interference prevent the acquisition of high-purity fungal DNA, thus seriously affecting the accuracy of subsequent fungal molecular biology analyses.

[0006] To achieve the objectives of this invention, the present invention is implemented through the following technical solution: a method for enriching fungi from the gastrointestinal contents of ruminants, comprising the following steps: Step 1: Collect fresh contents from different parts of the gastrointestinal tract of ruminants and screen the target parts with the highest fungal abundance by fungal-specific qPCR analysis; Step 2: Physically sieve the contents of the selected target area, then remove large particle impurities by vacuum filtration, and collect the precipitate by centrifugation; Step 3: The precipitate collected by centrifugation is resuspended in a compound enzyme digestion solution for overnight digestion. The compound enzyme digestion solution contains cellulase, chitinase, pectinase, glucanase, lysozyme, cell wall lysing enzyme, and DNase I (deoxyribonuclease I), which is used to dissociate the binding between fungi and plant materials, digest complex polysaccharides outside fungal cells, degrade free DNA, and partially lyse bacteria. Step 4: Centrifuge the overnight digested sample using a sucrose density gradient to collect fungal cells from the target concentration layer; Step 5: Resuspend the collected fungal cell pellet in PBS buffer containing lysozyme and digest overnight to further lyse the remaining bacteria. Then add DNase I and continue digestion for 1 hour. Centrifuge to collect the pellet and complete the deep purification. Step Six: Verify the enrichment effect of the collected fungal cells to complete the fungal enrichment.

[0007] A further improvement is made in the following: In step one, the ruminant is a dairy cow, and the different parts of the gastrointestinal tract include the reticulum, rectum, colon, cecum, abomasum, ileum, omasum, small intestine, and rumen. The relative abundance of fungi in each part is compared, and the target part with the highest fungal abundance is the abomasum. The fungal-specific qPCR analysis uses the ITS gene as a specific marker gene.

[0008] A further improvement is made in the following step: In step two, during physical sieving, the contents of the target area are suspended and stirred with PBS buffer (phosphate buffer) to ensure uniform dispersion. The contents are then filtered sequentially using sieves of different pore sizes, from largest to smallest, to remove large particles of feed residue and other physical impurities. The filtered solution is allowed to stand, and the supernatant is collected. Vacuum filtration is performed sequentially using 150μm and 100μm filter membranes to retain target fungal cells (such as spores and hyphal fragments) while allowing smaller bacteria to pass through. The material retained on the filter membrane or the concentrated filtrate is collected, centrifuged, and the precipitate is collected and washed with PBS. The centrifugation parameters for collecting the precipitate are 5000 rpm for 3 minutes.

[0009] A further improvement is made in the following: In step three, the overnight digestion temperature is 37°C, and the amount of cellulase, chitinase, pectinase, and glucanase added to the compound enzyme digestion solution is 0.2 g / 10 mL, and the amount of lysozyme, lysozyme, and DNase I added is 200 μL / 10 mL; or the amount of cellulase, chitinase, pectinase, and glucanase added is 0.5 g / 10 mL, and the amount of lysozyme, lysozyme, and DNase I added is 500 μL / 10 mL.

[0010] A further improvement is made in step four, when performing sucrose density gradient centrifugation, a discontinuous density gradient is constructed from bottom to top using 60%, 45%, 30%, and 15% sucrose solutions. The sample digested overnight is added to the top layer of the gradient solution and then placed in a horizontal rotor centrifuge and centrifuged at 12,000 rpm for 10 minutes. The target concentration layers are 30%, 45%, and 60% sucrose concentration layers, and the 15% sucrose concentration layer is discarded.

[0011] A further improvement is made in step five, where the centrifugation parameters for collecting the precipitate are 3000 rpm for 5 minutes, and the collected precipitate is flash-frozen in liquid nitrogen and then stored at -80°C.

[0012] A further improvement is made in step six, where the specific steps for verifying the enrichment effect are as follows: DNA is extracted from the original sample before enrichment, the sample after enrichment, and the deeply purified sample, respectively. After diluting the DNA to the same concentration, PCR amplification and qPCR analysis are performed using ITS as primers. The enrichment effect is verified by comparing the intensity and specificity of the PCR bands and the changes in the Cq value of qPCR.

[0013] Further improvements are made as follows: The PCR amplification system is as follows: Mix 5 μL, ITS primer 0.3 μL, DNA template 0.5 μL, ddH2O 4.2 μL, total system 10 μL. The amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 53℃ annealing for 10 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min, and storage at 4℃.

[0014] Further improvements are made in that the qPCR analysis system is consistent with the PCR amplification system, and the analysis procedure is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 53℃ annealing for 10 s and reading fluorescence, 72℃ extension for 30 s, 60 cycles; 72℃ final extension for 5 min, 4℃ incubation for 5 min.

[0015] The application of a method for enriching fungi from the gastrointestinal contents of ruminants in the preparation of a kit for analyzing fungal communities from the gastrointestinal contents of ruminants.

[0016] The application of a method for enriching fungi from the gastrointestinal contents of ruminants in the analysis, quantitative detection, or functional genomics research of fungal diversity in the gastrointestinal tract of ruminants, including fungal diversity analysis, quantitative detection of fungi, and fungal functional genomics research.

[0017] The beneficial effects of this invention are as follows: Addressing the technical challenges of low fungal biomass, tight integration with plant substrates, and severe bacterial background interference, this invention achieves highly efficient and specific enrichment of fungal cells through a combined strategy of physical sieving, complex enzymatic digestion, and density gradient centrifugation. Experiments have shown that the Cq value of the fungal ITS gene in the enriched sample decreased by 6-7 cycles in qPCR detection, with an enrichment efficiency of over 100 times, significantly improving detection sensitivity and specificity. Furthermore, the overall operation process is standardized and highly reproducible, providing a high-quality DNA starting point for downstream fungal diversity analysis, quantitative detection, and functional genomics research. It has broad application prospects in ruminant nutrition research and feed additive development. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the PCR results of the sedimentation layer, 15%, 30%, 45%, and 60% layers in the embodiments of the present invention, with concentrations of 10, 20, 40, 60, 80, and 100 ng / µL, respectively. Figure 2 This is a schematic diagram of the PCR results when the concentration of abomasal DNA was diluted to 10, 20, 40, and 60 ng / µL in the embodiments of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that the technical means not described in detail in the following embodiments are all conventional means in the art, are not the key points of the invention, and will not be elaborated upon.

[0021] Example 1 This embodiment involves the screening and confirmation of target sampling sites, including the following steps: 1. Sample collection Fresh intestinal contents were collected from different parts of the cow, including the reticulum, rectum, colon, cecum, abomasum, ileum, omasum, small intestine, and rumen. The samples were stored at 4°C for later use.

[0022] 2. DNA extraction Take 500 μL of the contents from each fraction and place them in an EP tube. Add 1 mL of lysis buffer to each tube, incubate at 65°C for 4 hours, and centrifuge briefly for 5 minutes. Collect the supernatant and add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Mix thoroughly and centrifuge at 12000 rpm for 15 minutes. If there is a large amount of white precipitate in the middle layer after centrifugation, repeat this step. Collect the supernatant and add an equal volume of chloroform:isoamyl alcohol (24:1). Mix thoroughly and centrifuge at 12000 rpm for 15 minutes. Collect the supernatant and add 2 volumes of anhydrous ethanol. Mix thoroughly and place in a -20°C freezer for 30 minutes to precipitate. Centrifuge at 12000 rpm for 15 minutes. Discard the supernatant, wash twice with 75% ethanol, and centrifuge at 12000 rpm for 15 minutes. After drying the EP tube, add an appropriate amount of ddH2O and determine the DNA concentration.

[0023] 3. Fungal Abundance Analysis DNA stock solutions from each region were diluted 10-fold, 100-fold, and 1000-fold, respectively, and then subjected to routine PCR amplification using universal primers for the fungal ITS region. The PCR reaction mixture consisted of: 5 μL of 2×Taq PCR Mix, 0.3 μL each of forward and reverse primers (10 μM), 0.5 μL of template DNA, and ddH2O to a final volume of 10 μL. The reaction program was as follows: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 53°C annealing for 10 seconds, 72°C extension for 30 seconds, for 35 cycles; final extension at 72°C for 5 minutes; and storage at 4°C. The amplified products were detected by agarose gel electrophoresis.

[0024] 4. qPCR validation DNA samples from all sites were diluted to the same concentration (20 ng / μL) and analyzed by real-time quantitative PCR (qPCR). The qPCR reaction system was the same as that for conventional PCR. The reaction program was as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 53℃ annealing for 10 seconds (fluorescence signal acquisition), 72℃ extension for 30 seconds, for a total of 60 cycles; final extension at 72℃ for 5 minutes; storage at 4℃. Three technical replicates were set up for each sample, and the copy number of the fungal ITS gene in each site was calculated according to the standard curve.

[0025] 5. Results Analysis qPCR results showed significant differences in fungal abundance across different intestinal sites (see Table 1). The abomasum sample exhibited the lowest mean circulation threshold (Cq value) at 19.25, corresponding to an ITS gene copy number of 6530.90 copies / μL, significantly higher than other sites such as the rumen (2056.33 copies / μL) and reticulum (463.64 copies / μL). This result indicates that the abomasum is the most abundant site for fungi in the bovine gastrointestinal tract, thus making it the target sampling site for subsequent fungal enrichment.

[0026] Table 1. Quantitative fluorescence results of different parts of the dairy cow intestine. Example 2 This embodiment involves the pretreatment and preliminary enrichment of abomasal contents, including the following steps: 1. Sample collection and dispersion processing Collect fresh bovine abomasal contents and store at 4°C. Take an appropriate amount of contents into a beaker, add 4 times the volume of phosphate-buffered saline (PBS, pH 7.2–7.4), and stir thoroughly with a magnetic stirrer for 20 minutes to ensure uniform dispersion of the contents.

[0027] 2. Physical sieving The dispersed sample was filtered sequentially through sieves of different pore sizes: first, a sieve with a larger pore size (e.g., 500 μm) was used to remove coarse feed particles, followed by filtration through a sieve with a medium pore size (e.g., 250 μm) and then a sieve with a smaller pore size (e.g., 100 μm), and the filtrate was collected. The filtrate was allowed to stand for 20 minutes to allow some fine particles to settle.

[0028] 3. Vacuum filtration Take the supernatant after settling and filter it sequentially through 150μm and 100μm filter membranes under vacuum. Discard the filtrate and collect the substance retained on the filter membrane. If the filter membrane is severely clogged, replace it with a new one. Wash the collected substance with an appropriate amount of PBS buffer, transfer it to a centrifuge tube, centrifuge at 5000 rpm for 3 minutes, and collect the precipitate. Wash the precipitate twice with PBS buffer, centrifuge, and collect for later use.

[0029] Example 3 This embodiment involves complex enzyme digestion, including the following steps: 1. Preparation of compound enzyme digestion solution Accurately weigh 0.2g of cellulase, 0.2g of chitinase, 0.2g of pectinase, and 0.2g of dextranase, place them in a container, and add 200μL of lysozyme, 200μL of lysozyme, and 200μL of deoxyribonuclease I (DNase I) sequentially. Adjust the volume to 20mL with PBS buffer and mix thoroughly to obtain the complex enzyme digestion solution. The final concentration of the enzyme solution should be adjusted according to the actual sample volume.

[0030] 2. Sample digestion treatment Wash the precipitate collected in Example 2 once with PBS buffer, centrifuge at 5000 rpm for 3 minutes, and discard the supernatant. Add freshly prepared compound enzyme digestion solution to the precipitate and resuspend it thoroughly. Place the resuspended solution in a 37°C constant temperature shaker and digest overnight (14 hours) at 150 rpm to fully dissociate the fungal cells from the plant substrate and degrade free DNA and some bacterial cells in the sample.

[0031] Example 4 This embodiment describes the separation of fungi by density gradient centrifugation of samples digested with a complex enzyme, including the following steps: 1. Preparation of sucrose density gradient solution Prepare sucrose solutions with mass / volume percentages of 60%, 45%, 30%, and 15%, respectively, using PBS buffer as the solvent. The prepared sucrose solutions can be stored at 4°C for later use.

[0032] 2. Density gradient centrifugation Take a 50 mL sterile centrifuge tube and, using a long-necked pipette or syringe, slowly add sucrose solution sequentially from high to low concentration along the tube wall: first add 10 mL of 60% sucrose solution, then carefully add 10 mL of 45% sucrose solution on top, followed by 10 mL of 30% sucrose solution, and finally 5 mL of 15% sucrose solution. A clear interface should be maintained between each concentration layer. Add the sample solution digested with the complex enzyme in Example 3 to the top layer of the gradient solution, taking care not to disrupt the gradient interface. After balancing the centrifuge tube, place it in a horizontal rotor centrifuge and centrifuge at 12,000 rpm for 10 minutes.

[0033] 3. Target layer collection and washing After centrifugation, the sample was observed to be distributed in layers of different densities. The top layer of liquid and the 15% sucrose layer, which mainly contained bacteria and small particulate impurities, were discarded. Samples from the 30%, 45%, and 60% sucrose layers and their interfaces were collected using a pipette. The collected samples were combined, 4 volumes of PBS buffer were added, and the mixture was thoroughly mixed. The mixture was centrifuged at 12,000 rpm for 10 minutes, and the precipitate was collected. The precipitate was washed twice with PBS buffer to remove residual sucrose.

[0034] 4. Preliminary Results Verification Total DNA was extracted from a small amount of the washed precipitate. DNA samples were diluted to 10, 20, 40, 60, 80, and 100 ng / μL, and PCR amplification was performed using fungal ITS primers. Electrophoresis results were then analyzed. Figure 1 The results showed that clear target bands were amplified in samples with 30%, 45%, and 60% sucrose layers, while no band was amplified in the 15% sucrose layer sample. This result indicates that fungal cells are mainly enriched in layers with 30%–60% sucrose density, and the method of this invention can effectively separate fungi from bacteria and other impurities.

[0035] Example 5 To further improve the purity of fungal cells, this embodiment performs deep purification on samples collected by density gradient centrifugation, including the following steps: 1. Lysozyme digestion The fungal cell pellet collected in Example 4 was resuspended in PBS buffer containing a high concentration of lysozyme (final concentration 2 mg / mL) and digested overnight (14 hours) in a 37°C shaker to further lyse any remaining bacterial cells.

[0036] 2. DNase I digestion After lysozyme digestion, DNase I (final concentration 100 U / mL) was added to the sample, and digestion continued at 37°C for 1 hour to degrade any remaining free DNA in the solution. After digestion, the sample was centrifuged at 12,000 rpm for 10 minutes, the precipitate was collected, and washed three times with PBS buffer.

[0037] 3. Verification of purification effect DNA samples were extracted separately after lysozyme digestion and DNase I lysis (steps as in Example 1). The resulting concentrations were homogenized and diluted to 10, 20, 40, and 60 ng / µL for PCR analysis. Results showed that lysozyme digestion yielded better results. The digested solutions were then centrifuged at 3000 rpm for 5 min. Electrophoresis results were then obtained. Figure 2The results showed that the amplification bands of the deeply purified samples were brighter and clearer, and the non-specific bands were significantly reduced, indicating that deep purification treatment can further improve the purity and quality of fungal DNA.

[0038] Example 6 To verify the stability and repeatability of the method of the present invention, this embodiment uses an optimized scheme to process new samples.

[0039] 1. Sample collection and pretreatment Fresh contents of the abomasum of five dairy cows were collected again and mixed to serve as experimental material. The samples were dispersed, filtered, and vacuum filtered according to the method in Example 2, and the precipitate was collected.

[0040] 2. Complex enzyme digestion The collected precipitate was resuspended in a compound enzyme digestion solution. The amount of compound enzyme digestion solution was scaled up according to the method in Example 3: 0.5 g cellulase, 0.5 g chitinase, 0.5 g pectinase, 0.5 g glucanase, 500 μL lysozyme, 500 μL lysozyme, and 500 μL DNase I. The solution was brought to a final volume of 50 mL with PBS buffer and digested overnight at 37°C with shaking.

[0041] 3. Density gradient centrifugation and collection A sucrose density gradient (60%, 45%, 30%, 15%) was prepared according to the method in Example 4. The digested solution was added to the top layer of the gradient liquid, and the mixture was centrifuged at 12,000 rpm for 10 minutes. Samples from the 30%, 45%, and 60% sucrose layers were collected, combined, and thoroughly washed with PBS buffer.

[0042] 4. Deep purification and efficacy verification The combined washed samples were divided into two portions: one portion was used for direct DNA extraction (enriched sample); the other portion underwent lysozyme digestion and DNase I lysis according to the method in Example 5 before DNA extraction. Enriched, lysozyme-digested, and DNase I-lysed DNA were extracted separately, using the same method as in Example 1. The resulting concentrations were homogenized and diluted to 10, 20, 40, and 60 ng / µL for PCR analysis. Results showed that lysozyme digestion yielded better results. The digested solution was then centrifuged at 3000 rpm for 5 min; the supernatant was removed, and the precipitate was transferred to cryovials and flash-frozen in liquid nitrogen at -80°C for subsequent DNA extraction, PCR amplification, high-throughput sequencing, and other molecular biological analyses.

[0043] As can be seen from the above embodiments, the method for enriching fungi from the gastrointestinal contents of ruminants provided by the present invention, through a combination strategy of target site screening, physical sieving, complex enzyme digestion, density gradient centrifugation and optional deep purification, can efficiently and specifically enrich fungal cells, significantly reduce the interference of bacteria and background DNA, and provide a high-quality material basis for subsequent research on fungal community structure and function.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enriching fungi from the gastrointestinal contents of ruminants, characterized in that, Includes the following steps: Step 1: Collect fresh contents from different parts of the gastrointestinal tract of ruminants and screen the target parts with the highest fungal abundance by fungal-specific qPCR analysis; Step 2: Physically sieve the contents of the selected target area, then remove large particle impurities by vacuum filtration, and collect the precipitate by centrifugation; Step 3: Resuspend the precipitate collected by centrifugation in a compound enzyme digestion solution for overnight digestion; Step 4: Centrifuge the overnight digested sample using a sucrose density gradient to collect fungal cells from the target concentration layer; Step 5: Resuspend the collected fungal cell pellet in PBS buffer containing lysozyme for overnight digestion, then add DNase I and continue digestion for 1 hour. Centrifuge to collect the pellet and complete the deep purification. Step 6: Verify the enrichment effect of the collected fungal cells.

2. The method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, characterized in that: In step one, the ruminant is a dairy cow, and the different parts of the gastrointestinal tract include the reticulum, rectum, colon, cecum, abomasum, ileum, omasum, small intestine, and rumen.

3. The method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, characterized in that: In step two, during physical sieving, the contents of the target area are suspended and stirred with PBS buffer, and filtered sequentially using sieves of different pore sizes from large to small. The vacuum filtration is carried out sequentially using 150μm and 100μm filter membranes.

4. The method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, characterized in that: In step three, the complex enzyme digestion solution contains cellulase, chitinase, pectinase, glucanase, lysozyme, lysozyme and DNase I.

5. A method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, characterized in that: In step four, when performing sucrose density gradient centrifugation, a discontinuous density gradient is constructed from bottom to top using 60%, 45%, 30%, and 15% sucrose solutions. After adding the overnight digested sample to the top layer of the gradient solution, centrifuge at 12,000 rpm for 10 minutes. The target concentration layers are 30%, 45%, and 60% sucrose concentration layers, and the 15% sucrose concentration layer is discarded.

6. A method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, characterized in that: In step five, the centrifugation parameters for collecting the precipitate are 3000 rpm for 5 min, and the collected precipitate is flash-frozen in liquid nitrogen and stored at -80℃.

7. A method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, characterized in that: In step six, the specific steps for verifying the enrichment effect are as follows: extract DNA from the original sample before enrichment, the sample after enrichment, and the deeply purified sample respectively; dilute the DNA to the same concentration; perform PCR amplification and qPCR analysis using ITS as primers; and verify the enrichment effect by comparing the intensity and specificity of the PCR bands and the changes in the Cq value of qPCR.

8. The use of the method for enriching fungi from the gastrointestinal contents of ruminants according to any one of claims 1-7 in the preparation of a kit for analyzing fungal communities from the gastrointestinal contents of ruminants.

9. The method for enriching fungi from the gastrointestinal contents of ruminants according to any one of claims 1-7, for the analysis, quantitative detection, or functional genomics research of fungal diversity in the gastrointestinal tract of ruminants.

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