A traditional Chinese medicine microecological preparation for preventing or treating calf diarrhea, a preparation method and application thereof

Abstract

This invention discloses a traditional Chinese medicine (TCM) microecological preparation for the prevention or treatment of diarrhea in calves and yaks, its preparation method, and its application, belonging to the field of TCM veterinary medicine technology. The preparation comprises a freeze-dried extract of TCM and freeze-dried probiotic powder; wherein the freeze-dried extract of TCM is prepared from *Pulsatilla chinensis*, *Clematis chinensis*, *Swertia davidii*, *Scutellaria baicalensis*, and *Glycyrrhiza uralensis*; the freeze-dried probiotic powder contains *Lactobacillus* with the preservation number CCTCC NO: M 20252010. This invention also provides a preparation method for the preparation, including steps such as decoction and concentration of TCM, freeze-drying, freeze-drying of *Lactobacillus* culture, and mixing of the two. This TCM microecological preparation has significant preventive and therapeutic effects on diarrhea in calves and yaks caused by multidrug-resistant *Escherichia coli*, effectively regulating the intestinal flora structure, increasing the diversity of intestinal fungi α, increasing the relative abundance of Firmicutes and *Ackermania*, and inhibiting the abnormal proliferation of pathogenic fungi, providing a new technical solution for the green prevention and control of diarrhea in calves and yaks.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine veterinary medicine technology, specifically relating to a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks, its preparation method and application. Background Technology

[0002] Yaks are cold-resistant livestock unique to the Qinghai-Tibet Plateau and have significant economic and ecological value. However, due to the harsh natural environment of high altitude, low oxygen, and cold, coupled with the long-standing traditional extensive farming management model and an imperfect disease prevention and control system, yaks, especially calves, are susceptible to various diseases, with bacterial diarrhea being the most common and serious. Pathogenic Escherichia coli is one of the main pathogens causing diarrhea in calves, with high morbidity and mortality rates, resulting in severe economic losses to the livestock industry.

[0003] Currently, antibiotics are the primary treatment for diarrhea in yaks. While they can control the disease in the short term, the long-term and excessive use of antibiotics has led to the widespread emergence of multidrug-resistant strains. This not only reduces treatment effectiveness but also poses serious problems such as drug residues, environmental pollution, and intestinal flora imbalance, threatening livestock product safety and human health. Therefore, developing safe, efficient, and residue-free alternative prevention and control solutions has become a critical technical challenge that the yak farming industry urgently needs to address.

[0004] In recent years, traditional Chinese medicine (TCM) and probiotics have gradually become research hotspots in the field of animal disease prevention and treatment due to their advantages such as being natural, safe, highly effective, and lacking drug resistance. TCM compound formulas, through the synergistic effects of multiple components, multiple targets, and multiple pathways, possess multiple efficacy functions, including antibacterial and anti-inflammatory effects, immune regulation, and improvement of intestinal function. Probiotics, on the other hand, exert their beneficial effects through mechanisms such as competitive exclusion, production of antibacterial substances, and regulation of host immunity. Studies have shown that the combined use of TCM and probiotics can produce significant synergistic effects. However, current research on TCM probiotics for calf yak diarrhea is limited, with a lack of dedicated formulations, and the compatibility and synergistic mechanisms between TCM formulations and probiotic strains remain unclear.

[0005] Therefore, in response to the actual needs of diarrhea prevention and treatment in calves and yaks, and in light of the ecological environment characteristics of the Qinghai-Tibet Plateau, there is an urgent need to develop a traditional Chinese medicine microecological preparation with antibacterial, anti-inflammatory, and immunomodulatory effects, as well as an inhibitory effect on multidrug-resistant Escherichia coli. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks, its preparation method, and its application. The traditional Chinese medicine microecological preparation of the present invention exhibits significant effects in the prevention and treatment of diarrhea in calves and yaks: it can effectively alleviate diarrhea symptoms, regulate the structure of intestinal bacteria and fungi, promote the proliferation of beneficial bacteria such as Firmicutes and Akkermansia, inhibit the abnormal proliferation of pathogenic fungi, and repair intestinal flora imbalance; simultaneously, by reducing the level of pro-inflammatory factors, increasing the expression of anti-inflammatory factors, enhancing antioxidant enzyme activity, and reducing the accumulation of oxidative damage products, it improves the anti-inflammatory and antioxidant capacity of calves and yaks, and promotes improved growth performance.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks, the traditional Chinese medicine microecological preparation comprising freeze-dried extracts of traditional Chinese medicine and freeze-dried probiotic powder; The freeze-dried extract of the Chinese herbal medicine is prepared from the following Chinese herbal raw materials in parts by weight: 25-30 parts of Pulsatilla chinensis, 25-30 parts of Clematis chinensis, 30-40 parts of Swertia japonica, 25-30 parts of Scutellaria baicalensis, and 25-30 parts of Glycyrrhiza uralensis. The probiotic freeze-dried powder contains Lactobacillus (CCTCC NO: M 20252010) Lactobacillus sp. YH02), viable count ≥2.5×10 10 CFU / g; of which, this lactobacillus ( Lactobacillus sp.YH02 was deposited on September 12, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0008] Preferably, the *Lactobacillus* with accession number CCTCC NO: M 20252010 has at least one of the following characteristics: (a) a survival rate ≥60% after treatment at 37°C for 3 hours in an environment with pH 3.0~5.0 and 0.1%~0.4% ox bile salts; (b) its fermentation supernatant exhibits resistance to multidrug-resistant *Escherichia coli* (M. 2025-2025). Escherichia coli The diameter of the inhibition zone is ≥12 mm.

[0009] Preferably, the mass ratio of the freeze-dried Chinese medicine extract to the freeze-dried probiotic powder is 1:(0.5~2).

[0010] This invention also provides a method for preparing the above-mentioned traditional Chinese medicine microecological preparation, comprising the following steps: (1) Mix Pulsatilla chinensis, Clematis armandii, Swertia japonica, Scutellaria baicalensis and Glycyrrhiza uralensis according to the above-mentioned weight ratio, add water and decoct 1 to 3 times, combine the decoctions, filter, concentrate to a relative density of 1.10 to 1.20 at 60℃, freeze dry, and obtain freeze-dried extract of Chinese medicine. (2) Lactobacillus with preservation number CCTCC NO: M 20252010 was inoculated into MRS medium and cultured at 37℃ for 8-10 h. The bacterial cells were collected, mixed with skim milk powder, and then freeze-dried to obtain probiotic freeze-dried powder. (3) Mix the freeze-dried extract of the Chinese herbal medicine with the freeze-dried powder of probiotics to obtain the Chinese herbal microecological preparation.

[0011] Preferably, in step (1), the addition of water for decoction refers to adding water in an amount of 8 to 12 times the total weight of the medicinal materials for decoction.

[0012] Preferably, in step (2), the mass concentration of the skim milk powder is 8%~15%; the mass ratio of the lactobacillus cells to the skim milk powder is 1:(1~3).

[0013] This invention also discloses the use of the above-mentioned traditional Chinese medicine microecological preparations in the preparation of drugs for the prevention or treatment of diarrhea in calves and yaks.

[0014] Preferably, the diarrhea in the calves is caused by multidrug-resistant Escherichia coli (M. coli). Escherichia coli )cause.

[0015] This invention also provides the use of the above-described traditional Chinese medicine microecological preparations in regulating the intestinal flora of calves and yaks, wherein the regulation includes at least one of the following: (a) Increase the diversity of gut fungi α; (b) Increase the relative abundance of Firmicutes and / or Akkermansia; (c) Reduce the relative abundance of Bacteroidetes; (d) Inhibit the abnormal proliferation of at least one pathogenic fungus selected from the genera Aspergillus, Fusarium, and Cercospora.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improves the growth performance of calves and yaks: The traditional Chinese medicine microecological preparation of the present invention can effectively promote the weight gain of calves and yaks, reduce the incidence of diarrhea, and improve the economic benefits of breeding.

[0017] (2) Effectively relieves inflammatory response: The traditional Chinese medicine microecological preparation of the present invention can significantly reduce the expression level of pro-inflammatory factors (such as TNF-α, IL-6, IL-1β, etc.) induced by Escherichia coli infection, while upregulating the level of anti-inflammatory factors such as IL-10, effectively reducing intestinal mucosal inflammation and damage, maintaining the integrity of the intestinal mucosal barrier, and reducing the risk of secondary infection.

[0018] (3) Enhance the body's antioxidant capacity: The traditional Chinese medicine microecological preparation of the present invention can significantly increase the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in calves and yaks, effectively remove excess reactive oxygen free radicals, reduce the accumulation of oxidative damage products such as malondialdehyde (MDA), significantly reduce oxidative stress damage, and enhance the body's immune defense function.

[0019] (4) Precise regulation of intestinal fungal community structure: The traditional Chinese medicine microecological preparation of the present invention can effectively inhibit the excessive proliferation of potential opportunistic pathogenic fungi (such as Cercospora, Aspergillus, Fusarium, etc.) caused by Escherichia coli infection, while promoting the enrichment of beneficial fungal groups (such as Xenodidymella, Hermatomyces, etc.) and restoring the ecological balance of the intestinal fungal community.

[0020] (5) Effectively regulates the composition of intestinal bacterial community: The traditional Chinese medicine microecological preparation of the present invention can significantly reduce the abundance of abnormally increased Bacteroides, Faecousia, Pararaprevotella and other conditionally pathogenic bacteria in the model group, while promoting the proliferation of bacteria with potential probiotic functions such as Rombutz and Akkermania, so as to restore the intestinal bacterial community structure to a healthy state.

[0021] (6) Maintaining intestinal microecological homeostasis: Through bidirectional regulatory effects, this preparation can effectively reverse intestinal flora disorder caused by Escherichia coli infection, promote the restoration of the Firmicutes / Bacteroidetes ratio to normal, and enhance the stability and anti-interference ability of the intestinal microecological system. Attached Figure Description

[0022] Figure 1 This image shows the colony morphology and Gram staining results of Lactobacillus.

[0023] Figure 2 The diagram shows the functional characteristics of Lactobacillus, where: A is the growth curve; B is the acid resistance; C is the bile salt resistance; and D is the antibacterial effect.

[0024] Figure 3 The graph shows the effects of traditional Chinese medicine microecological preparations on the growth performance and health indicators of calves and yaks. In the graph: A represents body weight; B represents diarrhea rate; C represents inflammation level; and D represents antioxidant capacity.

[0025] Figure 4 The figure shows the results of alpha diversity analysis of gut fungal communities in calves and yaks, where: A is the rarefaction curve; B is the rank abundance curve; and C is the alpha diversity indices.

[0026] Figure 5The figure shows the results of β-diversity analysis of the gut fungal community of calves and yaks, where: A is non-metric multidimensional scaling analysis (NMDS); B is unweighted group average clustering heatmap analysis (UPGMA).

[0027] Figure 6 A bar chart showing the species distribution of gut fungi in calves and yaks at different taxonomic levels, where: A represents the phylum level; B represents the genus level.

[0028] Figure 7 This figure shows the phylogenetic analysis results of the intestinal fungal flora of three groups of calves and yaks at the phylum level.

[0029] Figure 8 This figure shows the results of a marker genus-level difference analysis of the gut fungal flora in three groups of calves and yaks.

[0030] Figure 9 The figure shows the results of alpha diversity analysis of gut bacteria in calves and yaks, where: A is the rarefaction curve; B is the rank abundance curve; and C is the alpha diversity indices.

[0031] Figure 10 The figure shows the results of β-diversity analysis of gut bacterial flora in calves and yaks, where: A is principal coordinate analysis (PCoA); B is non-metric multidimensional scaling analysis (NMDS); and C is unweighted group average clustering heatmap analysis (UPGMA).

[0032] Figure 11 A bar chart showing the species distribution of gut bacteria in calves at different taxonomic levels, where: A represents the phylum level; B represents the genus level.

[0033] Figure 12 The figure shows the results of the indicative differences in gut bacterial flora at the phylum and genus levels in three groups of calves and yaks, where: A represents the phylum level; B represents the genus level. Detailed Implementation

[0034] 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.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] This invention provides a method for preparing a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks, the specific steps of which are as follows: (1) Take 25-30 parts of Pulsatilla chinensis, 25-30 parts of Clematis chinensis, 30-40 parts of Swertia japonica, 25-30 parts of Scutellaria baicalensis, and 25-30 parts of Glycyrrhiza uralensis. Mix them together and add water in 8-12 times the total weight of the herbs. Decoction 1-3 times, combine the decoctions, filter, concentrate to a relative density of 1.10-1.20 at 60℃, cool and freeze dry in a freeze dryer to obtain freeze-dried extract of Chinese medicine. Store in a sealed container.

[0038] (2) Lactobacillus with preservation number CCTCC NO: M 20252010 was inoculated into MRS medium and cultured at 37℃ for 8-10 h. The bacterial cells were collected. The bacterial cells were mixed with skim milk powder with a mass concentration of 8%-15% at a mass ratio of 1:(1-3), and freeze-dried to obtain probiotic freeze-dried powder. The viable count was ≥2.5×10⁻⁶. 10 CFU / g.

[0039] (3) Mix the freeze-dried extract of traditional Chinese medicine with the freeze-dried powder of probiotics at a mass ratio of 1: (0.5-2) to obtain a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks.

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments: Example 1 This example describes the isolation, identification, and characterization of Lactobacillus strains.

[0041] 1. Isolation and purification of bacterial strains Feces from healthy female yaks of the Jiangda yak population were collected and placed in 15 mL sterile centrifuge tubes. An appropriate amount of MRS liquid medium was added, and the mixture was initially cultured at 37°C. 200 μL of the initially cultured solution was then plated onto MRS solid medium and incubated at 37°C for 48 h. Lactic acid bacteria were isolated by serial dilution and plated again. The bacterial strain was purified by continuous streak plating to obtain pure cultures of single colonies. The colonies were smooth, milky-white spherical, and Gram staining showed short purple rods, no branching, and no spores (e.g., spores). Figure 1 (As shown).

[0042] 2. Molecular biological identification of the strain The purified strain was inoculated into 5 mL centrifuge tubes containing 3 mL of MRS medium and cultured overnight. Genomic DNA was then extracted using a kit. Molecular identification was performed using 16S rDNA PCR amplification: PCR amplification was performed on the genomic DNA extracted using the kit with universal primers. After preliminary analysis by agarose gel electrophoresis, samples meeting the expected size were sent to a professional company for sequencing. The sequencing results were analyzed using BLAST in the NCBI database, identifying the bacterium as *Lactobacillus*. Lactobacillus sp.). This strain is classified as... Lactobacillus sp. YH02 was deposited on September 12, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252010. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0043] 3. Characterization of strains (1) Determination of the growth characteristics of the strain: The *Lactobacillus* was inoculated into a 10 mL sterile test tube containing 5 mL of MRS medium and incubated at 37 °C. Samples were taken at 0 h, 2 h, 4 h, 5 h, 6 h, 7 h, 8 h, 10 h, 11 h, and 12 h to determine the OD value. 600 Values, plot growth curves, such as Figure 2 As shown in Figure A, the results indicate that the bacteria enters the stable phase after 9 hours, suggesting that its suitable fermentation cycle is 8 hours.

[0044] (2) Determination of the strain's acid and bile salt tolerance: The strains were inoculated into MRS liquid medium with pH adjusted to 2.0, 3.0, 4.0, and 5.0, as well as into MRS liquid medium with ox bile salt concentrations of 0, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%. After incubation at 37°C for 3 hours, the cultures were serially diluted 10-fold to 10⁻⁶. -6 100 μL of bacterial suspensions at various concentrations were spread onto MRS agar plates and incubated at 37°C for 24 h before colony counting. The results showed that the survival rate of this *Lactobacillus* was ≥60% after treatment at 37°C for 3 h in pH 3.0–5.0 and 0.1%–0.4% ox bile salt environments, indicating good acid and bile salt tolerance (e.g., ...). Figure 2 (As shown in B and 2C).

[0045] (3) Determination of the antibacterial activity of the strain: The Oxford cup method was used to determine the antibacterial activity, with ciprofloxacin susceptibility testing discs (20 μg / disc) used as a positive control. Multidrug-resistant Escherichia coli from yak was also tested. Escherichia coli (NCBI login number: PP859186) adjusted to 1×10 8CFU / mL, 100 μL was evenly spread onto the surface of an MRS agar plate. This *E. coli* strain was initially isolated from the feces of diarrheal yaks in Changdu, Tibet, and preserved in the Traditional Chinese Veterinary Laboratory of Nanjing Agricultural University. It exhibits multidrug resistance and is one of the representative pathogens causing diarrhea in high-altitude yaks. 100 μL of centrifuged *Lactobacillus* fermentation supernatant was added to each Oxford cup. Antimicrobial susceptibility testing discs were affixed to the blank area of ​​the Oxford cup plate after sample addition. The plates were incubated at 37℃ for 24 h, and the diameter of the inhibition zone was observed and measured.

[0046] The results showed that the fermentation supernatant of this *Lactobacillus* exhibited significant antibacterial activity against multidrug-resistant *Escherichia coli* with an inhibition zone diameter ≥20 mm (e.g., *Lactobacillus* fermentation supernatant). Figure 2 (as shown in D).

[0047] Example 2 This example describes the preparation of a traditional Chinese medicine microecological preparation.

[0048] The preparation method of the traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks in this embodiment is as follows: (1) Take 28 parts of Pulsatilla chinensis, 28 parts of Clematis armandii, 35 parts of Swertia japonica, 28 parts of Scutellaria baicalensis, and 28 parts of Glycyrrhiza uralensis. Mix them together and add water in a volume 10 times the total weight of the herbs. Decoction twice (2 hours the first time and 1.5 hours the second time). Combine the decoctions, filter, concentrate to a relative density of 1.15 at 60°C, cool and freeze dry in a freeze dryer (pre-freezing temperature -40°C, vacuum degree 10Pa, sublimation drying for 24 hours) to obtain the freeze-dried extract of Chinese medicine. Store in a sealed container.

[0049] (2) Lactobacillus with preservation number CCTCC NO: M 20252010 was inoculated into MRS medium and cultured at 37℃ for 9 h. The bacterial cells were collected by centrifugation at 4000 rpm for 10 min. The bacterial cells were mixed with 10% skim milk powder at a mass ratio of 1:2 and freeze-dried (pre-freezing temperature -40℃, vacuum degree 10 Pa, sublimation drying for 24 h) to obtain probiotic freeze-dried powder with a viable count of 2.5 × 10⁻⁶. 10 CFU / g.

[0050] (3) Mix the freeze-dried extract of traditional Chinese medicine with the freeze-dried powder of probiotics at a mass ratio of 1:1 to obtain a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks.

[0051] Example 3 This embodiment evaluates the therapeutic effect of the traditional Chinese medicine microecological preparation of the present invention on diarrhea in calves and yaks caused by multidrug-resistant Escherichia coli, and its promoting effect on the growth performance of calves and yaks. The specific experimental design and results are as follows: 1. Experimental materials Experimental animals: 30 healthy 2-month-old yaks from Leiwuqi County, Changdu City, Tibet Autonomous Region, with similar weights (average 18.5±1.2 kg) and good physical condition.

[0052] Pathogenic strain: Multidrug-resistant Escherichia coli from yak ( Escherichia coli (NCBI accession number: PP859186), originally isolated from the feces of diarrheal yaks in Changdu, Tibet, and preserved in the Traditional Chinese Veterinary Laboratory of Nanjing Agricultural University, exhibits a multidrug-resistant phenotype and is one of the representative pathogens of diarrhea in plateau yaks.

[0053] Traditional Chinese medicine microecological preparation: The traditional Chinese medicine microecological preparation prepared according to Example 2.

[0054] Control drug: physiological saline.

[0055] 2. Animal grouping and modeling The 30 calves were randomly divided into 3 groups of 10 each, and the experimental period was 15 days.

[0056] Control group (FCL): Normally fed, not challenged with the virus, and administered 10 mL of physiological saline orally daily; Model group (FML): 2×10⁻⁶ orally administered on day 1 of the experiment. 10 CFU-resistant E. coli was treated with 10 mL of physiological saline orally daily. Treatment group (FOL): 2×10⁻⁶ orally on day 1 of the experiment. 10 For CFU-resistant multidrug-resistant E. coli, administer 10 mL of physiological saline orally daily; simultaneously, administer a traditional Chinese medicine probiotic preparation (dosage: 0.5 g / kg BW) every three days.

[0057] 3. Analysis of experimental results: (1) Weight changes: such as Figure 3 As shown in Figure A, on day 15 of the experiment, the average weight of calves in the model group (FML) was significantly lower than that in the control group (FCL) (P<0.001); the average weight of calves in the treatment group (FOL) was significantly higher than that in the model group (FML) (P<0.01), and there was no significant difference between them and the control group (FCL) (P>0.05). This indicates that the traditional Chinese medicine microecological preparation prepared in this invention can effectively promote the growth and development of calves with diarrhea.

[0058] (2) Diarrhea symptoms: such as Figure 3As shown in Figure B, the model group (FML) began to experience diarrhea on day 1 after oral administration of E. coli, with a diarrhea rate of approximately 70%; the diarrhea rate was approximately 80% on days 2-9; and remained at approximately 70% on days 10-15. The treatment group (FOL) also experienced diarrhea on day 1, with a diarrhea rate of approximately 70% on days 1-2; approximately 60% on days 3-5; approximately 30% on days 6-9; and decreasing to approximately 10% on days 10-15, significantly lower than the model group (FML).

[0059] The results showed that the traditional Chinese medicine microecological preparation of the present invention can effectively treat diarrhea in calves and yaks caused by multidrug-resistant Escherichia coli, significantly improve diarrhea symptoms, and promote the recovery of growth performance in calves and yaks.

[0060] Example 4 This embodiment evaluates the regulatory effect of the traditional Chinese medicine microecological preparation of the present invention on the serum inflammatory factor levels and antioxidant capacity of diarrheal calves and yaks.

[0061] 1. Experimental materials Experimental animals: 30 calves of yaks and their groups as described in Example 3.

[0062] Traditional Chinese medicine microecological preparation: The traditional Chinese medicine microecological preparation prepared according to Example 2.

[0063] Main reagents: ELISA kit (for detecting IL-6, IL-1β, TNF-α, and IL-10), SOD, T-AOC, GSH-PX, and MDA detection kits.

[0064] 2. Sample collection Based on Example 3 above, on the 15th day of the experiment, 5 mL of jugular vein blood was collected from each group of calves and yaks in Example 3, placed in a sterile centrifuge tube, centrifuged at 12000 r / min for 15 min, the serum was separated, aliquoted and stored in a -80℃ refrigerator for later use.

[0065] 3. Analysis of Experimental Results (1) Serum inflammation level: like Figure 3As shown in Figure C, the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the serum of calves in the model group (FML) were significantly higher than those in the control group (FCL) and the treatment group (FOL), indicating that E. coli challenge successfully induced a systemic inflammatory response. After intervention with traditional Chinese medicine microecological preparations, the levels of the above three pro-inflammatory factors (IL-1β, TNF-α, and IL-6) in calves in the treatment group (FOL) were significantly lower than those in the model group (FML), suggesting that the preparation has a clear anti-inflammatory effect. Regarding anti-inflammatory factors, the IL-10 level in the model group (FML) was significantly lower than that in the control group (FCL) and the treatment group (FOL), while the IL-10 level in the treatment group (FOL) was close to that in the control group (FCL), indicating that traditional Chinese medicine microecological preparations can effectively enhance the expression of anti-inflammatory factors and achieve partial regulation and balanced repair of the inflammatory response.

[0066] This demonstrates that the traditional Chinese medicine microecological preparation of the present invention can significantly inhibit excessive inflammation activation caused by pathogen infection, while promoting anti-inflammatory pathway response, thus exhibiting synergistic immunomodulatory function.

[0067] (2) Serum antioxidant capacity: like Figure 3 As shown in Figure D, the serum antioxidant capacity of calves in the model group (FML) was significantly impaired, and the activities of antioxidant enzymes—superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-PX)—were significantly lower than those in the control group (FCL) and the treatment group (FOL). At the same time, the content of malondialdehyde (MDA), the lipid peroxidation end product, in the model group (FML) was significantly higher than that in the control group (FCL) and the treatment group (FOL), indicating that the body was in a state of severe oxidative stress.

[0068] After treatment with traditional Chinese medicine probiotics, the antioxidant capacity of the calves in the treatment group (FOL) was significantly improved. The activities of SOD, T-AOC and GSH-PX were significantly increased, while the MDA content was significantly reduced, which was close to that of the control group (FCL). Their overall level has obviously approached a healthy state.

[0069] The above results indicate that the traditional Chinese medicine microecological preparation of the present invention can effectively activate the endogenous antioxidant defense system of calves and reduce oxidative damage caused by pathogen infection.

[0070] Example 5 This embodiment evaluates the regulatory effect of the traditional Chinese medicine microecological preparation of the present invention on the structure and diversity of intestinal fungal flora in diarrheal calves and yaks.

[0071] 1. Experimental materials Experimental animals: 30 calves of yaks and their groupings as described in Example 3. Traditional Chinese medicine probiotic preparations: Traditional Chinese medicine probiotic preparations prepared according to Example 2 Main reagents: universal primers for the fungal ITS region ITS1F / ITS2, DNA extraction kit, PCR amplification reagents Main instrument: Illumina MiSeq high-throughput sequencing platform 2. Sample collection Based on Example 3 above, on the 15th day of the experiment, 5 mL of jugular vein blood was collected from each group of calves and yaks in Example 3, placed in a sterile centrifuge tube, centrifuged at 12000 r / min for 15 min, the serum was separated, aliquoted and stored in a -80℃ refrigerator for later use.

[0072] Total DNA was extracted from fecal samples using the CTAB method, and PCR amplification was performed using universal primers for the fungal ITS region (ITS1F / ITS2). The amplified products were purified and then subjected to PE300 paired-end sequencing using the Illumina MiSeq platform. The obtained raw sequences were analyzed for quality control, OTU clustering, α-diversity, β-diversity, and species annotation using QIIME2 software, and differential bacterial community markers were screened using LEfSe software.

[0073] 3. Analysis of Experimental Results (1) Sequencing data quality assessment: like Figure 4 As shown in A and 4B, the dilution curves of all samples gradually flatten out with increasing sequencing depth, indicating that the amount of sequencing data is reasonable and can fully demonstrate the diversity and richness of the fungal community.

[0074] (2) α-diversity analysis: like Figure 4 As shown in Figure C, the Shannon and Simpson indices of intestinal fungi in the model group (FML) calves were significantly lower than those in the control group (FCL); the Shannon and Simpson indices of the treatment group (FOL) were significantly higher than those in the model group (FML) (P<0.05), and there was no significant difference between the treatment group (FOL) and the control group (FCL) (P>0.05). This indicates that traditional Chinese medicine microecological preparations can significantly improve the α-diversity of intestinal fungi in diarrheal calves.

[0075] (3) β-diversity analysis: like Figure 5 As shown in Figure A, NMDS analysis revealed that the gut fungal flora of the three groups of calves was relatively dispersed, indicating significant differences in the flora structure among the groups. Figure 5As shown in B, the control group (FCL) and treatment group (FOL) samples clustered into the same main branch with high branch support, indicating that their microbial community compositions were highly similar. In contrast, the model group (FML) samples clustered into a separate branch, with a significantly increased distance from the former two, reflecting a significant deviation in their microbial community structure and indicating a large difference in their microbial community structure.

[0076] (3) Analysis of microbial community composition: Door horizontal: such as Figure 6 As shown in Figure A, the dominant phyla of intestinal fungi in the three groups of calves and yaks were Ascomycota, Fungiphy Incertae sedis, and Basidiomycota. The abundance of Ascomycota and Basidiomycota in the model group (FML) was significantly lower than that in the control group (FCL), while the abundance of Fungiphy Incertae sedis was significantly higher than that in the control group (FCL). Compared with the model group (FML), the abundance of Ascomycota and Basidiomycota in the treatment group (FOL) was significantly increased, while the abundance of Fungiphy Incertae sedis was significantly decreased.

[0077] Horizontal: such as Figure 6 As shown in Figure B, the main gut fungi in calves and yaks in the control group (FCL) and model group (FML) were *Thelebolus*, *Preussia*, and *Fungi gen Incertae sedis*, while those in the treatment group were mainly *Preussia*, *Podospora*, and *Fungi gen Incertae sedis*. The abundance of *Fungigen Incertae sedis* in the treatment group was significantly lower than that in the model group.

[0078] The results indicate that the traditional Chinese medicine microecological preparation of the present invention can effectively regulate the intestinal fungal community structure of calves and yaks, reverse the imbalance caused by Escherichia coli infection, especially promote the colonization of beneficial fungi such as the genera *Gnaphalium* and *Gnaphalium*, and restore the normal abundance of *Fungi gen Incertae sedis*, thereby enhancing the stability of the intestinal microecology and the host's anti-infection ability.

[0079] (4) Differential bacterial genera analysis: Door horizontal: such as Figure 7As shown, the relative abundance of Ascomycota and Basidiomycota in the intestines of calves in the model group (FML) was significantly lower than that in the control group (FCL), while the abundance of Neocallimastigomycota was significantly increased. After intervention with traditional Chinese medicine microecological preparations, the abundance of Ascomycota and Basidiomycota in the treatment group (FOL) recovered to levels close to those of the control group (FCL), while the abundance of Neocallimastigomycota was significantly reduced (P<0.05, marked with an asterisk in the figure).

[0080] The results indicate that the traditional Chinese medicine microecological preparation of the present invention can effectively reverse the imbalance of the dominant fungal phylum caused by Escherichia coli infection and promote the recovery of the intestinal fungal community to a healthy state.

[0081] Horizontal: such as Figure 8 As shown, there were significant differences in the gut fungal community structure among the groups, as detailed below: ① Comparison between the control group (FCL) and the treatment group (FOL): The relative abundance of *Thelebolus* in the FCL group was significantly higher than that in the FOL group (P<0.05); while the abundance of *Truncatella*, *Protomyces*, *Penicillium*, *Neostagonospora*, *Sporormia*, *Pseudocoleophoma*, *Malassezia*, and *Chrysosporium* in the FOL group was significantly higher than that in the FCL group (P<0.05 or P<0.01).

[0082] ② Comparison between the model group (FML) and the control group (FCL): The relative abundance of Rachicladosporium, Paraaleptosphaeria, Dioszegia, Vishniacozyma, Cercospora, Comoclathris, Aspergillus, Fusarium, Ramularia, Caecomyces, Cryptococcus, Eremomyces, Leptosphaeria, Lycoperdon, Sclerostagonospora and Bhatiellae in the FML group was significantly higher than that in the FCL group (P<0.05, P<0.01 or P<0.001).

[0083] ③ Therapeutic effect: After treatment with traditional Chinese medicine microecological preparations, the relative abundance of several genera (including Rachicladosporium, Dioszegia, Vishniacozyma, Cercospora, Ramularia, Caecomyces, Cryptococcus, Eremomyces, Lycoperdon, Sclerostagonospora and Bhatiellae) enriched in the FML group of calves in the treatment group (FOL) decreased to varying degrees.

[0084] Furthermore, the abundance of Xenodidymella, Dictyosporiaceaegen Incertae_sedis, Hermatomyces, and Mycocentrospora in the FOL group was significantly higher than that in the FCL and FML groups (P<0.05 or P<0.01). The abundance of Beauveria, Lapidomyces, and Heterocephalacria in the FML group was also significantly higher than that in the FCL and FOL groups (P<0.05 or P<0.01).

[0085] In summary, intervention with traditional Chinese medicine microecological preparations can effectively regulate the disordered intestinal fungal flora structure caused by Escherichia coli infection, inhibit the abnormal proliferation of potential pathogenic fungi (such as Aspergillus, Fusarium, Cercospora, etc.), and promote the enrichment of specific beneficial or symbiotic fungal groups, thereby promoting the recovery of the intestinal flora of calves and yaks to a healthy state.

[0086] Example 6 This embodiment evaluates the regulatory effect of the traditional Chinese medicine microecological preparation of the present invention on the intestinal bacterial flora structure of diarrheal calves and yaks.

[0087] 1. Experimental materials Experimental animals: 30 calves of yaks and their groups as described in Example 3; Traditional Chinese medicine microecological preparations: Traditional Chinese medicine microecological preparations prepared using Example 2; Main reagents: Universal primers 338F / 806R for bacterial 16S rRNA V3-V4 region, DNA extraction kit, PCR amplification reagents; Main instrument: Illumina MiSeq high-throughput sequencing platform.

[0088] 2. Sample collection On day 15 of the experiment, approximately 5 g of fresh fecal samples were collected from each group of calves and yaks. The samples were immediately placed in sterile centrifuge tubes, flash-frozen in liquid nitrogen, and then transferred to a -80°C freezer for storage (same batch as in Example 5).

[0089] Total DNA was extracted from fecal samples using the CTAB method. PCR amplification was performed using universal primers for the V3-V4 region of bacterial 16S rRNA. The amplified products were purified and then subjected to Illumina MiSeq PE300 paired-end sequencing. QIIME2 software was used for sequence quality control, OTU clustering, α-diversity, β-diversity, and species annotation analysis. LEfSe software was used to screen for differential bacterial community markers.

[0090] 3. Analysis of Experimental Results (1) Sequencing data quality assessment: like Figure 9 As shown in A and 9B, the dilution curves of all samples tend to flatten out as the sequencing depth increases, indicating that the sequencing data is sufficient to fully reflect the diversity and richness of the bacterial community in the samples.

[0091] (2) α-diversity analysis: like Figure 9 As shown in C, the α-diversity analysis results showed that there were no significant differences in any of the indicators among the groups (P>0.05), indicating that the Escherichia coli infection model group (FML) and the traditional Chinese medicine microecological preparation intervention treatment group (FOL) did not have a significant impact on the overall richness and diversity of the intestinal bacterial community of calves and yaks.

[0092] (3) β-diversity analysis: like Figure 10 A, Figure 10 B Figure 10 As shown in C, Principal Coordinate Analysis (PCoA) Figure 10 A) Non-metric multidimensional scaling (NMDS) Figure 10 B) Unweighted Group Average (UPGMA) Cluster Analysis Figure 10 C) The results consistently showed that the three groups of samples exhibited significant separation in their community structure. The control group (FCL) and treatment group (FOL) samples were relatively close to each other, while the model group (FML) samples were significantly separated. This indicates that E. coli infection significantly alters the intestinal bacterial community structure, and treatment with traditional Chinese medicine probiotics can, to some extent, reverse this change, restoring the bacterial community structure to a healthy state.

[0093] (3) Analysis of microbial community composition: Door horizontal: such as Figure 11As shown in Figure A, there were significant differences in the dominant bacterial phyla among the groups. The control group (FCL) was dominated by Firmicutes A (41.99%) and Proteobacteria (8.95%). The model group (FML) and treatment group (FOL) were dominated by Firmicutes A (FML=47.06%, FOL=46.14%) and Bacteroidetes D (FML=1.31%, FOL=8.84%), while the model group (FML=49.01%, FOL=29.12%) were the dominant phyla. *Escherichia coli* infection led to a significant decrease in Firmicutes D abundance and an increase in Bacteroidetes abundance. After treatment with traditional Chinese medicine probiotics, Firmicutes D abundance recovered, while Bacteroidetes abundance significantly decreased, and the bacterial composition returned to the control group (FCL) level.

[0094] Horizontal: such as Figure 11 As shown in B, the dominant bacterial genera varied among the three groups. In the control group (FCL), the dominant genera were *Acinetobacter* (29.27%), *Romboutsia* (13.37%), and *Faecousia* (4.62%). In the model group (FML), the dominant genera were *Bacteroides* (21.98%), *Faecousia* (19.62%), and *Paraprevotella* (13.78%). In the treatment group (FOL), the dominant genera were *Bacteroides* (16.83%), *Akkermansia* (9.24%), and *Romboutsia* (7.27%). After treatment with traditional Chinese medicine probiotics, the abundance of Bacteroides, Faecousia and Pararaprevotella, which were abnormally high in the treatment group (FOL), decreased, while the abundance of Rombutz and Akkermania, which have potential probiotic functions, increased significantly.

[0095] (4) Analysis of differentially expressed microbial biomarkers: Door horizontal: such as Figure 12As shown in Figure A, the relative abundance of Bacteroidota in the control group (FCL) was significantly lower than that in the model group (FML, P<0.001) and the treatment group (FOL, P<0.05); the relative abundance of FirmicutesD in the model group (FML) was significantly lower than that in the control group (FCL, P<0.05) and the treatment group (FOL, P<0.05); and the relative abundance of Actinobacteriota in the control group (FCL) was significantly higher than that in the model group (FML, P<0.05).

[0096] Horizontal: such as Figure 12 As shown in B, the relative abundance of Bacteroides H, Parabacteroides B 862066, Faecalimonas, Butyricicoccus A 77030, Dorea A, Frisingicoccus, Coprobacter, UBA2212, and Faecalibacterium in the model group (FML) was significantly higher than that in the control group (FCL); while the relative abundance of YIM 78166, Pradoshia, and Rhodococcus C 375578 was relatively low.

[0097] Furthermore, the relative abundance of Faecousia, Butyricimonas, Enterenecus, Bariatricus, and Oribacterium in the model group (FML) was significantly higher than that in the control group (FCL) and the treatment group (FOL); the relative abundance of Arthrobacter B in the control group (FCL) was significantly higher than that in the treatment group (FOL), while the relative abundance of Anaerotignum 189125 was significantly lower than that in the treatment group (FOL); the relative abundance of Clostridium T in the treatment group (FOL) was significantly higher than that in the model group (FML), and the relative abundance of Stercorousia in the control group (FCL) was significantly higher than that in both the model group (FML) and the treatment group (FOL); (P<0.05 or P<0.01).

[0098] After treatment with traditional Chinese medicine probiotics, the relative abundance of Bacteroides H, Faecalimonas, Dorea A, Frisingicoccus, Coprobacter, and Faecalibacterium, which were abnormally high in the model group, decreased to varying degrees in the treatment group (FOL), while the relative abundance of YIM 78166, Pradoshia, and Rhodococcus C 375578 increased. Simultaneously, the relative abundance of Faecousia, Butyricimonas, Enterenecus, Bariatricus, and Oribacterium in the treatment group (FOL) decreased significantly, approaching the levels of the control group (FCL).

[0099] In summary, the traditional Chinese medicine microecological preparation of the present invention can effectively regulate the intestinal bacterial flora structure disorder caused by Escherichia coli infection, inhibit the abnormal proliferation of specific bacterial genera, and promote the enrichment of beneficial bacterial genera, thereby promoting the restoration of the intestinal microecological system to a healthy state.

[0100] The foregoing has provided a detailed description of a traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these examples are merely illustrative of the methods and core ideas of the invention. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims.

Claims

1. A traditional Chinese medicine microecological preparation for the prevention or treatment of diarrhea in calves and yaks, characterized in that, The traditional Chinese medicine microecological preparation includes freeze-dried extracts of traditional Chinese medicine and freeze-dried probiotic powder; The freeze-dried extract of the Chinese herbal medicine is prepared from the following Chinese herbal raw materials in parts by weight: 25-30 parts of Pulsatilla chinensis, 25-30 parts of Clematis chinensis, 30-40 parts of Swertia japonica, 25-30 parts of Scutellaria baicalensis, and 25-30 parts of Glycyrrhiza uralensis. The probiotic freeze-dried powder contains Lactobacillus with preservation number CCTCC NO: M 20252010 ( Lactobacillus sp. YH02), viable count ≥ 2.5 × 10⁻⁶ 10 CFU / g.

2. The traditional Chinese medicine microecological preparation according to claim 1, characterized in that, The *Lactobacillus* with accession number CCTCC NO: M20252010 possesses at least one of the following characteristics: (a) a survival rate ≥60% after treatment at 37°C for 3 hours in an environment with pH 3.0–5.0 and 0.1%–0.4% bovine bile salts; (b) its fermentation supernatant exhibits resistance to multidrug-resistant *Escherichia coli* (M. spp.). Escherichia coli The diameter of the inhibition zone is ≥12 mm.

3. The traditional Chinese medicine microecological preparation according to claim 1 or 2, characterized in that, The mass ratio of the freeze-dried Chinese medicine extract to the freeze-dried probiotic powder is 1:(0.5~2).

4. A method for preparing a traditional Chinese medicine microecological preparation as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Pulsatilla chinensis, Clematis armandii, Swertia japonica, Scutellaria baicalensis and Glycyrrhiza uralensis are mixed in the weight ratio described in claim 1, decocted with water 1 to 3 times, the decoctions are combined, filtered, concentrated to a relative density of 1.10 to 1.20 at 60°C, and freeze-dried to obtain a freeze-dried extract of traditional Chinese medicine. (2) Lactobacillus with preservation number CCTCC NO: M 20252010 was inoculated into MRS medium and cultured at 37℃ for 8-10 h. The bacterial cells were collected, mixed with skim milk powder, and then freeze-dried to obtain probiotic freeze-dried powder. (3) Mix the freeze-dried extract of the Chinese herbal medicine with the freeze-dried powder of probiotics to obtain the Chinese herbal microecological preparation.

5. The preparation method according to claim 4, characterized in that, In step (1), adding water for decoction means adding 8 to 12 times the total weight of the medicinal materials for decoction.

6. The preparation method according to claim 4, characterized in that, In step (2), the mass concentration of the skim milk powder is 8% to 15%; the mass ratio of the lactobacillus cells to the skim milk powder is 1:(1 to 3).

7. The use of the traditional Chinese medicine microecological preparation according to any one of claims 1-3 in the preparation of a drug for the prevention or treatment of diarrhea in calves and yaks.

8. The use according to claim 7, characterized in that, The diarrhea in the calves was caused by multidrug-resistant Escherichia coli (M. coli). Escherichia coli )cause.

9. The use of the traditional Chinese medicine microecological preparation according to any one of claims 1-3 in regulating the intestinal flora of calves and yaks, characterized in that, The adjustment includes at least one of the following: (a) Increase the diversity of gut fungi α; (b) Increase the relative abundance of Firmicutes and / or Akkermansia; (c) Reduce the relative abundance of Bacteroidetes; (d) Inhibit the abnormal proliferation of at least one pathogenic fungus selected from the genera Aspergillus, Fusarium, and Cercospora.

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