A veterinary composition for the treatment of diarrhea

Abstract

The application discloses a veterinary composition for preventing and treating diarrhea, and relates to the technical field of veterinary drugs.The composition is composed of a constipating and detoxicating group, a qi-enriching and spleen-strengthening group and a heat-clearing and dampness-drying group, the first two groups are anaerobically fermented by specific lactobacillus to prepare a bifunctional compound fermentation liquor, the heat-clearing and dampness-drying group is prepared into an extract by low-temperature flash extraction, and then the two groups are mixed in a specific manner to prepare oral liquid preparations or drinking water powders loaded by modified attapulgite adsorption.The veterinary composition prepared by the application has high retention rate of key active ingredients, synergistic effects of multiple action mechanisms, remarkable prevention and treatment effects on diarrhea of livestock and poultry, and can replace antibiotics to prevent and treat diarrhea of livestock and poultry in large-scale breeding.

Description

Technical Field

[0001] This invention relates to the field of veterinary drug technology, specifically to a veterinary drug composition for the prevention and treatment of diarrhea and dysentery. Background Technology

[0002] Diarrhea in livestock and poultry is one of the diseases with the highest morbidity and mortality rates in large-scale farming, especially harming weaned piglets and young poultry. Statistics show that approximately 70% of deaths in piglets under 60 days of age are related to diarrhea. The causes of diarrhea are complex, including infection by pathogens such as enterotoxigenic Escherichia coli, stimulation by feed antigens, and incomplete development of digestive function. For a long time, livestock production has relied mainly on antibiotics for prevention and treatment. However, the long-term and excessive use of antibiotics has led to increased bacterial resistance, excessive drug residues in animal products, and intestinal flora imbalance. Against this backdrop, using traditional Chinese medicine and probiotics to replace antibiotics has become an important development direction in the veterinary drug field.

[0003] In the prior art, there are various schemes that combine traditional Chinese medicine with probiotics for the prevention and treatment of diarrhea in livestock and poultry. For example, patent application publication number CN112586595B discloses a traditional Chinese medicine microecological compound preparation for the prevention and treatment of enteritis in newborn piglets, as well as its preparation method and application. This scheme combines Astragalus membranaceus, Rhizoma Scutellariae Radix et Rhizoma, Taraxaci Herba, Radix Isatidis, and Folium Artemisiae Argyi in proportion by weight to form a compound traditional Chinese medicine. After boiling with water, the extract is concentrated to obtain a traditional Chinese medicine extract. Then, a mixed bacterial solution of Lactobacillus plantarum and Lactobacillus amyloliquefaciens is added for anaerobic fermentation to produce a compound preparation. As another example, patent application publication number CN116210820A discloses a traditional Chinese medicine fermented feed for the prevention and treatment of diarrhea in piglets and its preparation method. This scheme mixes multiple traditional Chinese medicines (including Astragalus membranaceus, Radix Paeoniae Alba, Fructus Psoraleae, Fructus Crataegi, Fructus Lycii, Rhizoma Mentha, and Radix Dioscoreae) with Bacillus subtilis, Lactobacillus, Bacillus licheniformis, yeast, and substrate, and ferments them in one step. However, the aforementioned existing technologies all employ a method of mixing and fermenting all Chinese herbal medicine components with probiotics in the same system in a single process, without addressing the differences in the chemical properties of the various Chinese herbal medicine components. Especially for compound Chinese herbal formulas used to treat diarrhea and dysentery, the alkaloids in heat-clearing and dampness-drying herbs (such as Coptis chinensis and Phellodendron chinense) exhibit poor stability in the acidic environment of prolonged fermentation, while the tannins and other macromolecular active ingredients in astringent and laxative herbs (such as Terminalia chebula and pomegranate peel) require sufficient enzymatic hydrolysis for effective release. Co-fermenting both in the same system leads to the dilemma of insufficient tannin release and significant alkaloid degradation losses. Furthermore, the role of probiotics in these schemes is mainly manifested in utilizing their enzymatic system to degrade macromolecular Chinese herbal medicines to improve the dissolution rate of active ingredients, and utilizing the metabolites of probiotics (such as organic acids) to exert a certain antibacterial effect. They do not involve designing methods to pre-combine probiotic metabolites with specific active ingredients of Chinese herbal medicines during preparation to form new material structures with synergistic effects.

[0004] The technical problem this invention aims to solve is that existing methods for preparing veterinary traditional Chinese medicine compositions for treating diarrhea and dysentery, which involve the one-time mixed fermentation of different functional components, fail to simultaneously ensure the full enzymatic release of tannins and the stable protection of heat- and acid-sensitive alkaloids. This results in the loss of active ingredients and a lack of effective synergistic effects among the components. The solution to this problem cannot be achieved simply by combining existing technologies, as there is no guidance or inspiration in the prior art to group traditional Chinese medicine compound prescriptions according to their functions, process each group separately using targeted methods, and then combine them sequentially.

[0005] There is an urgent need for a veterinary drug composition and its preparation method that can be differentiated based on the chemical characteristics and functional differences of traditional Chinese medicine components, so that the active ingredients of different functional components can be retained and utilized to the greatest extent, and multiple mechanisms of action can be effectively synergistically achieved at the formulation level, thereby improving the antidiarrheal effect and reducing the loss of active ingredients. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a veterinary drug composition for the prevention and treatment of diarrhea. By grouping traditional Chinese medicine compound according to its function and chemical properties and processing it with a differentiated method, the tannin components are fully released and form supramolecular complexes with the metabolites of probiotics. At the same time, the active ingredients of alkaloids are effectively protected, and the synergistic effect of multiple mechanisms of action is achieved, thereby significantly improving the prevention and treatment effect of diarrhea in livestock and poultry. It can replace antibiotics for large-scale breeding.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On one hand, a veterinary drug composition for the prevention and treatment of diarrhea and dysentery, the composition comprising an astringent and antidiarrheal group, a qi-tonifying and spleen-strengthening group, and a heat-clearing and dampness-drying group: the astringent and antidiarrheal group comprises 60-90 parts by weight of Terminalia chebula, 40-70 parts by weight of pomegranate peel, and 30-50 parts by weight of Sanguisorba officinalis; the qi-tonifying and spleen-strengthening group comprises 50-80 parts by weight of Astragalus membranaceus, 40-60 parts by weight of Codonopsis pilosula, and 30-50 parts by weight of Atractylodes macrocephala; the heat-clearing and dampness-drying group comprises 20-40 parts by weight of Coptis chinensis, 20-40 parts by weight of Phellodendron chinense, and 30-60 parts by weight of Pulsatilla chinensis.

[0008] The astringent and detoxifying group and the qi-tonifying and spleen-strengthening group are first subjected to anaerobic fermentation by a lactobacillus strain with anti-enterotoxin-producing Escherichia coli activity. This process causes the condensed tannins and hydrolyzable tannins in the astringent and detoxifying group to be degraded and released by the fermentation enzyme system. After being released, they form a tannin-probiotic metabolite supramolecular complex with bacteriocins and surface proteins produced by the extracellular metabolism of the lactobacillus strain through non-covalent binding. At the same time, the astragalus polysaccharides and codonopsis saponins in the qi-tonifying and spleen-strengthening group are fully dissolved, thereby obtaining a bifunctional complex fermentation broth. This bifunctional complex fermentation broth has the ability to neutralize bioactive toxins from the metabolites of the lactobacillus strain, the ability to neutralize chemical toxins from the tannins in the astringent and detoxifying group, and the ability to repair mucosal tissue and regulate immunity from the qi-tonifying and spleen-strengthening group.

[0009] The heat-clearing and dampness-drying group was separately extracted by low-temperature flash extraction to obtain a heat-clearing and dampness-drying extract. Berberine, palmatine alkaloids, and Pulsatilla saponins in the heat-clearing and dampness-drying extract were retained under the condition that the material temperature did not exceed 30°C.

[0010] The bifunctional complex fermentation broth and the heat-clearing and drying extract are mixed under stirring conditions by a fine stream injection. In the acidic environment formed by the bifunctional complex fermentation broth, the alkaloids in the heat-clearing and drying extract are stably dissolved in the form of salts.

[0011] The composition contains berberine at a content of not less than 0.8 mg / mL, tannins at a content of not less than 2.0 mg / mL (calculated as gallic acid), astragalus polysaccharide at a content of not less than 1.5 mg / mL (calculated as glucose), and a pH value of 4.0–5.5.

[0012] By combining the astringent and detoxifying group, the qi-tonifying and spleen-strengthening group, and the heat-clearing and dampness-drying group in specific weight ratios, and by subjecting the astringent and detoxifying group and the qi-tonifying and spleen-strengthening group to anaerobic fermentation with a strain of lactobacillus exhibiting activity against enterotoxin-producing Escherichia coli, the condensed tannins and hydrolyzable tannins in the astringent and detoxifying group are fully degraded and released under the action of fermentation enzymes. These tannins then non-covalently bind with bacteriocins and surface proteins produced by the extracellular metabolism of the lactobacillus strain to form a supramolecular complex of tannins and probiotic metabolites. This supramolecular complex possesses both the bioactive toxin neutralizing ability derived from lactobacillus metabolites and the chemical toxin neutralizing ability derived from the tannins in the astringent and detoxifying group. Simultaneously, it allows the astragalus polysaccharides and codonopsis saponins in the qi-tonifying and spleen-strengthening group to be fully dissolved, providing mucosal repair and immune regulation. The heat-clearing and drying components are extracted separately using a low-temperature flash extraction method, and then mixed with the fermentation broth of the bifunctional complex under stirring conditions using a fine-stream injection method. This allows the berberine and palmatine alkaloids in the heat-clearing and drying extract to be retained at a material temperature not exceeding 30°C. They also dissolve stably in the form of salts in the acidic environment formed by the fermentation broth of the bifunctional complex, avoiding the precipitation of alkaloids and tannins due to acid-base neutralization, which would cause both to become ineffective. The resulting composition contains no less than 0.8 mg / mL of berberine, no less than 2.0 mg / mL of tannins (calculated as gallic acid), no less than 1.5 mg / mL of astragalus polysaccharide (calculated as glucose), and a pH value of 4.0–5.5, ensuring that the content of key active ingredients and the pH of the formulation are stable and controllable in the finished product.

[0013] On the other hand, a method for preparing a veterinary drug composition for the prevention and treatment of diarrhea, applicable to the veterinary drug composition for the prevention and treatment of diarrhea as described in claim 1, is characterized in that the method comprises the following steps:

[0014] Step 1: Mix all the medicinal materials of the astringent and detoxifying group (after purification and ultra-fine pulverization to 50μm-75μm) and all the medicinal materials of the qi-tonifying and spleen-strengthening group, add 8-10 times the weight of sterile distilled water, sterilize with flowing steam at 105℃ for 20 min, cool to 37±1℃, inoculate with seed liquid of Lactobacillus strain with anti-enterotoxin-producing Escherichia coli activity, the inoculation amount is 5% v / v of the total fermentation system volume, ferment at 37±1℃, anaerobic, initial pH 6.0-6.5, and stirring speed 30-50 rpm for 48-72 h to obtain bifunctional complex fermentation broth;

[0015] Step 2: Mix all the medicinal materials of the heat-clearing and dampness-drying group that have been cleaned and coarsely crushed to 2mm-4mm, add 20 times their weight of weakly acidic distilled water with pH 4.5-5.0, and flash extract at 20℃-30℃ and a speed of 8000rpm-12000rpm for 60s-90s. Repeat the extraction twice. The extract is first filtered through a 200-mesh sieve and then finely filtered through a 0.45μm microporous membrane to obtain the heat-clearing and dampness-drying extract.

[0016] Step 3: Under the conditions of 20℃~30℃ and stirring speed of 50rpm~80rpm, the heat-clearing and drying extract obtained in step 2 is injected into the bifunctional complex fermentation broth obtained in step 1 in a thin stream. The injection speed does not exceed 1 / 20 / min of the volume of the bifunctional complex fermentation broth obtained in step 1. After injection, the mixture is allowed to stand at room temperature for 4h~6h for maturation, and then filtered through a 0.45μm microporous membrane.

[0017] By first ultra-finely pulverizing the astringent and detoxifying group and the qi-tonifying and spleen-strengthening group and inoculating them with anti-enterotoxin lactobacillus for anaerobic fermentation, while simultaneously performing low-temperature flash extraction on the heat-clearing and dampness-drying group, and then sequentially mixing and maturing the two products at a controlled rate, the degradation of alkaloids in the acidic fermentation environment can be avoided, the full enzymatic release of tannins can be promoted, and they can be pre-combined with probiotic metabolites, thereby maximizing the retention of active ingredients and achieving synergistic effects.

[0018] Furthermore, the screening method for Lactobacillus strains with anti-enterotoxin-producing Escherichia coli activity in step 1 is as follows: enrichment culture and isolation purification are carried out in the colon contents of healthy adult pigs using MRS selective medium containing gradient concentrations of pomegranate peel tannins, and strains that can still grow under the condition of tannin concentration not less than 2.0 mg / mL and have the highest anti-enterotoxin activity against enterotoxin-producing Escherichia coli in the fermentation broth are screened.

[0019] By screening Lactobacillus strains from the colon contents of healthy adult pigs using MRS selective medium containing gradient concentrations of pomegranate peel tannins, which can still grow under conditions of tannin concentration not less than 2.0 mg / mL and have the highest activity against enterotoxin-producing Escherichia coli enterotoxins, a dedicated fermentation strain with both high tannin tolerance and strong enterotoxin resistance can be obtained, ensuring the directional formation of effective complex structures in the bifunctional complex fermentation broth.

[0020] Furthermore, the endpoint of fermentation in step 1 is determined as follows: the pH of the fermentation broth drops to 4.0±0.3, the tannin content increases by no less than 30% compared to the unfermented control water extract, and the soluble polysaccharide content increases by no less than 25%.

[0021] By using the fermentation broth pH dropping to 4.0±0.3, the tannin content increasing by no less than 30%, and the soluble polysaccharide content increasing by no less than 25% as the criteria for determining the fermentation endpoint, it is possible to ensure that the probiotic enzyme system achieves a balance between the degradation degree of Chinese medicine macromolecules and the accumulation of metabolites, so that the amount of bifunctional complex generated is in the optimal range.

[0022] Furthermore, in step 2, the weakly acidic distilled water is adjusted to pH 4.5–5.0 using food-grade citric acid, and the material temperature does not exceed 30°C during the extraction process.

[0023] By using food-grade citric acid-adjusted weakly acidic distilled water (pH 4.5–5.0) as the flash extraction solvent and controlling the material temperature during the extraction process to not exceed 30°C, alkaloids such as berberine in Coptis chinensis and Phellodendron amurense can be efficiently dissolved in salt form, while protecting heat-sensitive components such as Pulsatilla chinensis saponins from damage.

[0024] Furthermore, the particle size of the ultrafine pulverized medicinal material in step 1 is 50μm to 75μm, and the particle size of the coarsely pulverized medicinal material in step 2 is 2mm to 4mm.

[0025] By ultra-finely pulverizing the medicinal materials to be fermented to 50μm~75μm and coarsely pulverizing the flash extraction medicinal materials to 2mm~4mm, the requirements of cell wall disruption rate in the fermentation process and mechanical shearing efficiency in the flash extraction process can be met respectively, thereby improving the tannin release and alkaloid extraction rate.

[0026] Furthermore, step 3 prepares an oral liquid formulation.

[0027] By directly filtering the liquid after time-sequential mixing and maturation into an oral liquid preparation, a finished dosage form that can be administered directly through drinking water can be obtained, which facilitates group administration in large-scale breeding and reduces the cost of manual administration.

[0028] Furthermore, the preparation method further includes the following after step 3:

[0029] Step 4: The purified and acid-modified attapulgite clay is added to the fermentation broth of the bifunctional complex obtained in Step 1 at a dosage of 5% w / w to 8% w / w, at a temperature of 25℃ to 30℃ and a stirring speed of 60 rpm to 80 rpm. After adsorption for 4 h to 6 h, the mixture is centrifuged to obtain the loaded complex solid and the supernatant.

[0030] Step 5: Inject the heat-clearing and drying extract obtained in step 2 into the supernatant obtained in step 4 in a thin stream. The injection speed shall not exceed 1 / 20 / min of the volume of the supernatant obtained in step 4. After the injection is completed, a mixture is obtained.

[0031] Step 6: Add the solid load composite obtained in Step 4 to the mixture obtained in Step 5 and stir for 30 min. Dry under vacuum at 45±3℃ for 6 h to 8 h until the moisture content does not exceed 5%. Crush and pass through an 80-100 mesh sieve.

[0032] After step 3, purified and acid-modified attapulgite clay is added to the fermentation broth of the bifunctional complex for adsorption and loading. The supernatant is then sequentially mixed with the heat-clearing and drying extract. Finally, the loaded solid and the mixture are combined and dried under low temperature and vacuum to obtain the drinking powder. This method utilizes the layered chain structure of attapulgite to load the bifunctional complex as a sustained-release carrier, prolonging the action time of the active ingredients in the intestine. At the same time, the residual effective ingredients in the fermentation broth are recovered and reused, improving the utilization rate of raw materials and the stability of the formulation.

[0033] Furthermore, the preparation method of the purified and acid-modified attapulgite clay in step 4 is as follows: the raw attapulgite clay ore is slurried with water, allowed to settle, dried at 105℃, and ball-milled to 50μm~75μm. Then, it is treated with 1.5mol / L hydrochloric acid at a liquid-to-solid ratio of 6:1mL / g and at 60℃ for 3h. After washing with pure water until the pH of the washing solution is 6.5~7.0 and no chloride ions are detected, it is dried at 105℃ until the water content does not exceed 3%.

[0034] By slurrying, settling, drying, and ball milling the raw attapulgite clay ore, and then stirring it with 1.5 mol / L hydrochloric acid at a liquid-to-solid ratio of 6:1 mL / g and 60℃ for 3 h, followed by washing with pure water until the washing solution pH is 6.5-7.0 and no chloride ions are detected, and then drying, the carbonate cement in the pores can be removed and the number of surface silanol groups can be increased, resulting in a modified attapulgite carrier with significantly improved adsorption capacity and adsorption active sites.

[0035] Furthermore, the drinking powder prepared in step 6 contains at least 2.0 mg of berberine, at least 5.0 mg of tannin, at least 3.5 mg of astragalus polysaccharide per 1 g, with a water content not exceeding 5.0%, lead not exceeding 10 mg / kg, arsenic not exceeding 2 mg / kg, coliform bacteria not exceeding 10 CFU / g, and Salmonella not detectable.

[0036] By controlling the content of berberine (no less than 2.0 mg), tannin (no less than 5.0 mg), and astragalus polysaccharide (no less than 3.5 mg) per 1g of the drinking water powder, and specifying the limits for moisture content, heavy metals, and microorganisms, the content of key active ingredients in the finished product can be kept stable, ensuring the effectiveness and safety of the product.

[0037] Compared with existing technologies, this veterinary drug composition for the prevention and treatment of diarrhea has the following beneficial effects:

[0038] I. This invention divides traditional Chinese medicine compound into three groups based on their functions and chemical properties: an astringent and detoxifying group, a qi-tonifying and spleen-strengthening group, and a heat-clearing and dampness-drying group. Different groups are then treated using targeted methods and mixed in a specific sequence. The astringent and detoxifying group and the qi-tonifying and spleen-strengthening group are first subjected to anaerobic fermentation with probiotics, which promotes the full degradation and release of tannin macromolecules under the action of fermentation enzymes. Simultaneously, the heat-clearing and dampness-drying group is extracted separately using low-temperature flash extraction, which effectively avoids the degradation of alkaloids in the acidic fermentation environment. This solves the problem that existing whole-component mixed fermentation methods cannot simultaneously ensure the full release of tannins and the stable protection of alkaloids, significantly improving the retention rate of various key active ingredients in the preparation.

[0039] Second, the present invention designs a preparation method that allows probiotic metabolites and tannins from traditional Chinese medicine to pre-combine during the preparation process to form a supramolecular complex. This complex can simultaneously exert the dual effects of neutralizing biological toxins and chemical toxins. Furthermore, it forms a multi-mechanism synergy with the mucosal repair and immune regulation effects of the Qi-tonifying and spleen-strengthening components and the antibacterial effects of the heat-clearing and dampness-drying components, thus significantly improving the prevention and treatment of diarrhea in livestock and poultry.

[0040] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0042] Figure 1 This is a flowchart of the preparation method of the present invention;

[0043] Figure 2 This is a diagram illustrating the mechanism of action of the supramolecular complex of the present invention. Detailed Implementation

[0044] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0045] like Figure 1As shown, the preparation method mainly consists of three stages. The first stage is the preparation of the bifunctional complex fermentation broth, in which the herbs of the astringent and detoxifying group and the qi-tonifying and spleen-strengthening group are ultra-finely pulverized and then inoculated with a special lactobacillus for anaerobic fermentation. The second stage is the preparation of the heat-clearing and dampness-drying extract, in which the herbs of the heat-clearing and dampness-drying group are coarsely pulverized and then subjected to low-temperature flash extraction. The third stage is the formulation, in which the two extracts are mixed in a specific manner and can be directly made into an oral liquid, or made into a drinking powder after being adsorbed and loaded with attapulgite.

[0046] like Figure 2 As shown, the tannin-probiotic metabolite supramolecular complex formed in this invention uses bacteriocins and surface proteins produced during fermentation as its core framework, binding to condensed tannins and hydrolyzable tannin molecules released during degradation through hydrogen bonds and hydrophobic interactions. This structure can simultaneously neutralize bioactive enterotoxins produced by bacteria and chemical toxins in feed, and remains stable in the acidic environment of the intestine.

[0047] Example 1

[0048] This embodiment specifically discloses a method for preparing an oral liquid veterinary drug for the prevention and treatment of diarrhea and dysentery. The medicinal materials of each group are weighed according to a specific weight ratio. The herbs of the astringent and digestive-strengthening group and the qi-tonifying and spleen-strengthening group are ultra-finely pulverized and inoculated with anaerobic fermentation of *Escherichia coli* and *Lactobacillus* strains selected through screening. The fermentation endpoint parameters are controlled to obtain a bifunctional complex fermentation broth. The herbs of the heat-clearing and dampness-drying group are coarsely pulverized and subjected to low-temperature flash extraction to obtain a heat-clearing and dampness-drying extract. The heat-clearing and dampness-drying extract is slowly injected into the bifunctional complex fermentation broth in a thin stream, allowed to stand and mature, and then finely filtered to obtain the oral liquid preparation. The oral liquid preparation prepared in this embodiment has stable content of key active ingredients and meets the pH requirements, and can be directly administered to livestock and poultry through drinking water.

[0049] The specific implementation process is as follows:

[0050] First, screening for active Lactobacillus strains resistant to enterotoxin-producing Escherichia coli was conducted. 10g of colonic contents from healthy adult pigs was added to 90mL of sterile physiological saline, thoroughly shaken and mixed. After standing for 10 minutes, the supernatant was collected and serially diluted. 10g of the supernatant was then taken. -4 10 -5 10 -6 Three dilutions of bacterial suspension, 0.1 mL each, were spread onto MRS selective agar plates containing gradient concentrations of pomegranate peel tannins. The pomegranate peel tannin concentrations were set at 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, and 4.0 mg / mL, respectively. The plates were incubated in a 37°C anaerobic incubator for 48 h. Single colonies that grew well at a tannin concentration of at least 2.0 mg / mL were picked and subjected to multiple streak isolation and purification.

[0051] The purified strains were subjected to an enterotoxin assay against enterotoxin-producing *Escherichia coli*. Each strain was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 48 h. The supernatant was collected by centrifugation. The neutralizing ability of the supernatant against enterotoxin-producing *Escherichia coli* was determined using a cytotoxicity assay. A *Lactobacillus* strain exhibiting vigorous growth and the highest enterotoxin activity at a tannin concentration of 2.5 mg / mL was selected and prepared as a seed culture. The seed culture was prepared by inoculating the strain into MRS liquid medium and anaerobically culturing at 37°C for 18 h until the OD600 value reached 1.8–2.0.

[0052] Weigh out 75 parts by weight of Terminalia chebula, 55 parts by weight of pomegranate peel, and 40 parts by weight of Sanguisorba officinalis to form an astringent and prolapse-fixing group. Weigh out 65 parts by weight of Astragalus membranaceus, 50 parts by weight of Codonopsis pilosula, and 40 parts by weight of Atractylodes macrocephala to form a qi-tonifying and spleen-strengthening group. Clean the two groups of herbs separately to remove impurities and non-medicinal parts. Then mix the two groups of herbs evenly and pulverize them using an ultra-micro pulverizer, controlling the particle size to be 50μm~75μm. Take 10kg of the pulverized herbal mixture, add 80kg of sterile distilled water, stir evenly, and place it in a fermentation tank. Sterilize with flowing steam at 105℃ for 20min. After sterilization, circulate sterile cooling water to cool the temperature inside the fermentation tank to 37±1℃.

[0053] The initial pH of the fermentation system was adjusted to 6.0–6.5, and the prepared Lactobacillus seed culture was inoculated at 5% v / v of the total fermentation system volume. Fermentation was carried out at 37±1℃ under anaerobic conditions and with a stirring speed of 30–50 rpm. Samples were taken every 6 hours during fermentation to determine the pH, tannin content, and soluble polysaccharide content of the fermentation broth. Fermentation was stopped when the pH of the fermentation broth dropped to 4.0±0.3, the tannin content increased by at least 30% compared to the unfermented control water extract, and the soluble polysaccharide content increased by at least 25%. In this example, the fermentation time was 60 hours to obtain the bifunctional complex fermentation broth.

[0054] Weigh out 30 parts by weight of Coptis chinensis, 30 parts by weight of Phellodendron chinense, and 45 parts by weight of Pulsatilla chinensis to form a heat-clearing and dampness-drying group. Clean the above-mentioned medicinal materials separately to remove impurities and non-medicinal parts. Then, mix the three groups of medicinal materials evenly and pulverize them using a coarse pulverizer, controlling the particle size to 2mm-4mm. Take 5kg of the pulverized medicinal material mixture and add 100kg of weakly acidic distilled water adjusted to pH 4.5-5.0 with food-grade citric acid. Flash extract at 10000rpm for 75s at 20℃-30℃, repeating the extraction twice. Combine the two extracts, first filter through a 200-mesh sieve to remove the residue, then finely filter through a 0.45μm microporous membrane to obtain the heat-clearing and dampness-drying extract. During the extraction process, cooling water circulation is used to control the material temperature to not exceed 30℃.

[0055] Under conditions of 20℃~30℃ and stirring speed of 50rpm~80rpm, the heat-clearing and drying extract obtained above was slowly injected into the bifunctional complex fermentation broth in a thin stream. The injection rate was controlled to not exceed 1 / 20 / min of the bifunctional complex fermentation broth volume. In this example, the bifunctional complex fermentation broth volume was 85L, the heat-clearing and drying extract volume was 95L, the injection rate was controlled at 4L / min, and the total injection time was approximately 24min. After injection, stirring was stopped, and the mixture was allowed to stand at room temperature for 5h for maturation. After maturation, the mixture was filtered through a 0.45μm microporous membrane to obtain an oral liquid preparation.

[0056] This embodiment successfully prepared a veterinary oral liquid preparation for the prevention and treatment of diarrhea according to the technical solution of the present invention. Testing revealed that the oral liquid preparation contained 1.2 mg / mL berberine, 2.8 mg / mL tannins (calculated as gallic acid), 2.1 mg / mL as astragalus polysaccharide (calculated as glucose), and a pH of 4.6. All indicators met the requirements specified in this invention. The oral liquid preparation prepared in this embodiment can be directly added to the drinking water of livestock and poultry for group administration, making it convenient to use and suitable for the prevention and treatment of diarrhea in livestock and poultry in large-scale farming.

[0057] Example 2

[0058] This embodiment specifically discloses a method for preparing a veterinary drug powder for the prevention and treatment of diarrhea and dysentery. Based on the preparation of the bifunctional complex fermentation broth and the heat-clearing and drying extract in Example 1, purified and acid-modified attapulgite clay is used to adsorb and load the bifunctional complex. Centrifugation is then used to separate the loaded complex solid and the supernatant. The heat-clearing and drying extract is injected into the supernatant in a thin stream to obtain a mixture. The loaded complex solid is then added to the mixture and stirred until homogeneous. After low-temperature vacuum drying, pulverization, and sieving, the powder is obtained. The powder prepared in this embodiment has a high content of key active ingredients, good stability, and is easy to store and transport.

[0059] The specific implementation process is as follows:

[0060] First, purified and acid-modified attapulgite clay was prepared. 10 kg of raw attapulgite clay ore was added to 50 kg of purified water and slurried. After stirring for 30 minutes, the mixture was allowed to settle for 2 hours, and the bottom coarse sand and upper floating matter were discarded. The intermediate layer of slurry was removed and dried at 105℃ to constant weight. The dried attapulgite clay was then ball-milled to a particle size of 50 μm–75 μm.

[0061] Take 5 kg of ball-milled attapulgite clay and add 30 L of 1.5 mol / L hydrochloric acid. Stir for 3 h at a liquid-to-solid ratio of 6:1 mL / g and 60 °C. After treatment, centrifuge and discard the supernatant. Wash the precipitate repeatedly with purified water until the pH of the washing solution reaches 6.5–7.0 and no chloride ions are detected by silver nitrate solution. Dry the washed precipitate at 105 °C until the moisture content does not exceed 3%, obtaining purified and acid-modified attapulgite clay.

[0062] The bifunctional complex fermentation broth and the heat-clearing and drying extract were prepared using the same method as in Example 1. 100 L of the bifunctional complex fermentation broth was taken and 6 kg of the purified and acid-modified attapulgite clay prepared above was added. The mixture was stirred and adsorbed for 5 h at 25℃~30℃ and a stirring speed of 60 rpm~80 rpm. After adsorption was complete, centrifugation was performed at 3000 rpm for 15 min to obtain the loaded complex solid and the supernatant.

[0063] Under conditions of 20℃~30℃ and stirring speed of 50rpm~80rpm, the heat-clearing and drying extract was slowly injected into the supernatant obtained above in a thin stream. The injection rate was controlled to not exceed 1 / 20 of the supernatant volume / min. In this example, the supernatant volume was 92L, the heat-clearing and drying extract volume was 95L, the injection rate was controlled at 4.5L / min, and the total injection time was approximately 21min. After injection, stirring was stopped to obtain a mixture.

[0064] The above-obtained loaded composite solid was added to the mixture, and stirring was continued for 30 minutes to ensure full dispersion of the loaded composite solid. The mixture was then transferred to a vacuum drying oven and dried under vacuum at 45±3℃ for 7 hours until the moisture content of the material did not exceed 5%. The dried material was then removed, pulverized using a pulverizer, and passed through an 80-100 mesh sieve to obtain the drinking water powder.

[0065] This embodiment successfully prepared a veterinary drug drinking powder for the prevention and treatment of diarrhea and dysentery according to the technical solution of the present invention. Testing revealed that each 1g of the drinking powder contained 2.6mg of berberine, 6.2mg of tannins, 4.3mg of astragalus polysaccharide, a water content of 3.8%, a lead content of 4.2mg / kg, an arsenic content of 0.8mg / kg, and a coliform count of 3 CFU / g. Salmonella was not detected. All indicators met the requirements specified in the present invention. The drinking powder prepared in this embodiment utilizes the sustained-release effect of attapulgite, which can prolong the action time of the active ingredients in the intestine, improve the therapeutic effect, and facilitate storage and transportation.

[0066] Comparative Example

[0067] This comparative example uses a commonly used full-component mixed fermentation method to prepare an antidiarrheal veterinary drug formulation. All medicinal materials from the astringent and digestive groups, the qi-tonifying and spleen-strengthening group, and the heat-clearing and dampness-drying group were mixed and subjected to water extraction and fermentation together to compare the advantages of the differentiated treatment method of this invention. The results of this comparative example show that the full-component mixed fermentation method leads to a significant degradation and loss of alkaloid active ingredients, while the release of tannins is insufficient, resulting in a significantly lower content of key active ingredients in the formulation compared to the embodiments of this invention.

[0068] The specific implementation process is as follows:

[0069] Weigh out the same amounts of each group of medicinal materials as in Example 1. That is, 75 parts by weight of Terminalia chebula, 55 parts by weight of pomegranate peel, 40 parts by weight of Sanguisorba officinalis, 65 parts by weight of Astragalus membranaceus, 50 parts by weight of Codonopsis pilosula, 40 parts by weight of Atractylodes macrocephala, 30 parts by weight of Coptis chinensis, 30 parts by weight of Phellodendron chinense, and 45 parts by weight of Pulsatilla chinensis. Mix all the medicinal materials evenly, clean them, and then pulverize them using a coarse pulverizer to a particle size of 2 mm to 4 mm.

[0070] Take 15 kg of the pulverized herbal mixture, add 120 kg of sterile distilled water, stir well, and place in an extraction tank. Extract by boiling at 100℃ for 2 hours, then filter to obtain the first extract. Add another 80 kg of sterile distilled water to the residue, and extract by boiling at 100℃ for 1.5 hours, then filter to obtain the second extract. Combine the two extracts and concentrate to a clear paste with a relative density of 1.10–1.15.

[0071] The extract was transferred to a fermenter, and sterile distilled water was added to adjust the volume to 150 L. The mixture was sterilized by flowing steam at 105 °C for 20 min, and then cooled to 37 ± 1 °C. The initial pH was adjusted to 6.0–6.5, and the same Lactobacillus seed culture as in Example 1 was inoculated at 5% v / v of the total fermentation volume. Fermentation was carried out at 37 ± 1 °C under anaerobic conditions with a stirring speed of 30–50 rpm for 60 h. After fermentation, the mixture was filtered through a 0.45 μm microporous membrane to obtain the complete fermentation preparation.

[0072] This comparative example uses a conventional whole-component mixed fermentation method to prepare an antidiarrheal veterinary drug formulation. Testing revealed that the formulation contained berberine at 0.3 mg / mL, tannins (calculated as gallic acid) at 1.2 mg / mL, and astragalus polysaccharide (calculated as glucose) at 0.9 mg / mL, with a pH of 3.8. Compared to Example 1 of this invention, the berberine content decreased by 75%, the tannin content decreased by 57%, and the astragalus polysaccharide content decreased by 57%. The results indicate that the whole-component mixed fermentation method cannot simultaneously ensure the sufficient enzymatic release of large tannin molecules and the stable protection of acid-sensitive alkaloids, leading to a significant loss of active ingredients. This invention effectively solves the problems existing in the prior art by treating different functional components using differentiated methods.

[0073] To further verify the technical effects of the present invention, a comparative experiment was conducted on the efficacy of the preparations prepared in Examples 1, 2, and the comparative example in preventing and treating piglet diarrhea. 120 21-day-old weaned piglets of similar weight and good health were randomly divided into four groups of 30 piglets each: a blank control group, Example 1 group, Example 2 group, and a comparative example group.

[0074] The blank control group was given a basal diet and normal drinking water. In Example 1, the oral liquid formulation prepared in Example 1 of this invention was added to the drinking water at a rate of 2 mL per 1 L of water. In Example 2, the drinking water powder prepared in Example 2 of this invention was added to the drinking water at a rate of 1 g per 1 L of water. In the comparative control group, the comparative control group received a complete component mixed fermentation formulation prepared in the comparative control at a rate of 2 mL per 1 L of water. The experimental period was 14 days.

[0075] Specifically, during the experiment, the diarrhea rate of piglets in the blank control group was 46.7%, the diarrhea rate in the Example 1 group was 10.0%, the diarrhea rate in the Example 2 group was 6.7%, and the diarrhea rate in the comparative group was 26.7%. The results show that the formulation prepared in the examples of this invention has a significantly better preventive and therapeutic effect on piglet diarrhea than the comparative formulation. Among them, the drinking water powder prepared in Example 2 has the best preventive and therapeutic effect due to its sustained-release effect.

[0076] The following is a comparison table of key parameters and effects for Example 1, Example 2, and the comparative example:

[0077] Berberine content 1.2 mg / mL 2.6 mg / g 0.3 mg / mL Tannin content 2.8 mg / mL 6.2mg / g 1.2 mg / mL Astragalus polysaccharide content 2.1 mg / mL 4.3 mg / g 0.9 mg / mL pH value 4.6 - 3.8 Piglet diarrhea rate 10.0% 6.7% 26.7% Key Dosage Form Advantages Easy to use and suitable for group dosing Good stability and long duration of action The method is simple

[0078] In summary, this invention effectively resolves the contradiction between insufficient tannin release and alkaloid degradation loss in existing technologies by grouping traditional Chinese medicine compound prescriptions according to their functions, treating each group separately with targeted methods, and then combining them sequentially. The veterinary drug composition prepared by this invention has a high content of key active ingredients and a synergistic mechanism of action, exhibiting good preventive and therapeutic effects on livestock and poultry diarrhea. It can replace antibiotics for the prevention and treatment of livestock and poultry diarrhea in large-scale farming.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A veterinary drug composition for the prevention and treatment of diarrhea and dysentery, characterized in that, The composition consists of an astringent and prolapse-preserving group, a qi-tonifying and spleen-strengthening group, and a heat-clearing and dampness-drying group: the astringent and prolapse-preserving group consists of 60-90 parts by weight of Terminalia chebula, 40-70 parts by weight of pomegranate peel, and 30-50 parts by weight of Sanguisorba officinalis; the qi-tonifying and spleen-strengthening group consists of 50-80 parts by weight of Astragalus membranaceus, 40-60 parts by weight of Codonopsis pilosula, and 30-50 parts by weight of Atractylodes macrocephala; and the heat-clearing and dampness-drying group consists of 20-40 parts by weight of Coptis chinensis, 20-40 parts by weight of Phellodendron chinense, and 30-60 parts by weight of Pulsatilla chinensis. The astringent and detoxifying group and the qi-tonifying and spleen-strengthening group are first subjected to anaerobic fermentation by a lactobacillus strain with anti-enterotoxin-producing Escherichia coli activity. This process causes the condensed tannins and hydrolyzable tannins in the astringent and detoxifying group to be degraded and released by the fermentation enzyme system. After being released, they form a tannin-probiotic metabolite supramolecular complex with bacteriocins and surface proteins produced by the extracellular metabolism of the lactobacillus strain through non-covalent binding. At the same time, the astragalus polysaccharides and codonopsis saponins in the qi-tonifying and spleen-strengthening group are fully dissolved, thereby obtaining a bifunctional complex fermentation broth. This bifunctional complex fermentation broth has the ability to neutralize bioactive toxins from the metabolites of the lactobacillus strain, the ability to neutralize chemical toxins from the tannins in the astringent and detoxifying group, and the ability to repair mucosal tissue and regulate immunity from the qi-tonifying and spleen-strengthening group. The heat-clearing and dampness-drying group was separately extracted by low-temperature flash extraction to obtain a heat-clearing and dampness-drying extract. Berberine, palmatine alkaloids, and Pulsatilla saponins in the heat-clearing and dampness-drying extract were retained under the condition that the material temperature did not exceed 30°C. The bifunctional complex fermentation broth and the heat-clearing and drying extract are mixed under stirring conditions by a fine stream injection. In the acidic environment formed by the bifunctional complex fermentation broth, the alkaloids in the heat-clearing and drying extract are stably dissolved in the form of salts.

2. A method for preparing a veterinary composition for the prophylactic and therapeutic treatment of diarrhea, which is suitable for the veterinary composition for the prophylactic and therapeutic treatment of diarrhea according to claim 1, characterized in that, The method includes the following steps: Step 1: Mix all the medicinal materials of the astringent and detoxifying group (after purification and ultra-fine pulverization to 50μm-75μm) and all the medicinal materials of the qi-tonifying and spleen-strengthening group, add 8-10 times the weight of sterile distilled water, sterilize with flowing steam at 105℃ for 20 min, cool to 37±1℃, inoculate with seed liquid of Lactobacillus strain with anti-enterotoxin-producing Escherichia coli activity, the inoculation amount is 5% v / v of the total fermentation system volume, ferment at 37±1℃, anaerobic, initial pH 6.0-6.5, and stirring speed 30-50 rpm for 48-72 h to obtain bifunctional complex fermentation broth; Step 2: Mix all the medicinal materials of the heat-clearing and dampness-drying group that have been cleaned and coarsely crushed to 2mm-4mm, add 20 times their weight of weakly acidic distilled water with pH 4.5-5.0, and flash extract at 20℃-30℃ and a speed of 8000rpm-12000rpm for 60s-90s. Repeat the extraction twice. The extract is first filtered through a 200-mesh sieve and then finely filtered through a 0.45μm microporous membrane to obtain the heat-clearing and dampness-drying extract. Step 3: Under the conditions of 20℃~30℃ and stirring speed of 50rpm~80rpm, the heat-clearing and drying extract obtained in step 2 is injected into the bifunctional complex fermentation broth obtained in step 1 in a thin stream. The injection speed does not exceed 1 / 20 / min of the volume of the bifunctional complex fermentation broth obtained in step 1. After injection, the mixture is allowed to stand at room temperature for 4h~6h for maturation, and then filtered through a 0.45μm microporous membrane.

3. The method of claim 2, wherein the veterinary composition is prepared by mixing the active ingredients and the excipient in a ratio of 1: 1 to 1:

3. The screening method for Lactobacillus strains with anti-enterotoxin-producing Escherichia coli activity in step 1 is as follows: enrichment culture and isolation purification are carried out in the colon contents of healthy adult pigs using MRS selective medium containing gradient concentrations of pomegranate peel tannins. The strains that can still grow under the condition of tannin concentration not less than 2.0 mg / mL and have the highest anti-enterotoxin activity against enterotoxin-producing Escherichia coli in the fermentation broth are screened.

4. The method of claim 2, wherein the veterinary composition is prepared by mixing the active ingredients and the excipient in a ratio of 1: 1 to 1:

3. The endpoint of fermentation in step 1 is determined as follows: the pH of the fermentation broth drops to 4.0±0.3, the tannin content increases by no less than 30% compared to the unfermented control water extract, and the soluble polysaccharide content increases by no less than 25%.

5. The method of claim 2, wherein the veterinary composition is prepared by mixing the active ingredients with the pharmaceutically acceptable carrier. In step 2, the weakly acidic distilled water is adjusted to pH 4.5–5.0 using food-grade citric acid, and the material temperature does not exceed 30°C during the extraction process.

6. The method of claim 2, wherein the veterinary composition is prepared by mixing the active ingredients and the excipient. The particle size of the medicinal material in the ultrafine pulverization in step 1 is 50μm to 75μm, and the particle size of the medicinal material in the coarse pulverization in step 2 is 2mm to 4mm.

7. The method of claim 2, wherein the veterinary composition is prepared by mixing the active ingredients with the pharmaceutically acceptable carrier. Step 3 prepares an oral liquid formulation.

8. The method of claim 2, wherein the veterinary composition is prepared by mixing the active ingredients and the excipient. The preparation method further includes, after step 3: Step 4: The purified and acid-modified attapulgite clay is added to the fermentation broth of the bifunctional complex obtained in Step 1 at a dosage of 5% w / w to 8% w / w, at a temperature of 25℃ to 30℃ and a stirring speed of 60 rpm to 80 rpm. After adsorption for 4 h to 6 h, the mixture is centrifuged to obtain the loaded complex solid and the supernatant. Step 5: Inject the heat-clearing and drying extract obtained in step 2 into the supernatant obtained in step 4 in a thin stream. The injection speed shall not exceed 1 / 20 / min of the volume of the supernatant obtained in step 4. After the injection is completed, a mixture is obtained. Step 6: Add the solid load composite obtained in Step 4 to the mixture obtained in Step 5 and stir for 30 min. Dry under vacuum at 45±3℃ for 6 h to 8 h until the moisture content does not exceed 5%. Crush and pass through an 80-100 mesh sieve.

9. The method of claim 8, wherein the veterinary composition is prepared by mixing the active ingredients with the pharmaceutically acceptable carrier. The preparation method of purified and acid-modified attapulgite clay in step 4 is as follows: the raw attapulgite clay ore is slurried with water, allowed to settle, dried at 105℃, and ball-milled to 50μm~75μm. Then, it is treated with 1.5mol / L hydrochloric acid at a liquid-to-solid ratio of 6:1mL / g and at 60℃ for 3h. After washing with pure water until the pH of the washing solution is 6.5~7.0 and no chloride ions are detected, it is dried at 105℃ until the water content does not exceed 3%.

10. The method of claim 8, wherein the veterinary composition is prepared by mixing the active ingredients with the pharmaceutically acceptable carrier. The drinking powder prepared in step 6 contains at least 2.0 mg of berberine, at least 5.0 mg of tannin, at least 3.5 mg of astragalus polysaccharide per 1 g, with a water content not exceeding 5.0%, lead not exceeding 10 mg / kg, arsenic not exceeding 2 mg / kg, coliform bacteria not exceeding 10 CFU / g, and Salmonella not detectable.

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