Method for treating autism spectrum disorder and multi-layered dripping pill containing intestinal flora

Abstract

The present invention relates to a method for treating autism spectrum disorders and a multilayer dripping pill containing intestinal flora. On the one hand, the multilayer dripping pills involved include a multilayer (e.g., 2 to 5 layers) structure of inner layer, middle layer, and outer layer in a concentric circle shape, wherein: the inner layer is a homogeneous mixture of live microorganisms such as probiotics or bacterial powder of intestinal flora and oily substances; the middle layer is an oily substance; the outer layer is a rubber shell formed by dripping and drying a colloid formed by a polymer material, a plasticizer, and water. The multilayer dripping pills of the present invention are used to encapsulate biologically active substances, especially for encapsulating live probiotics or fecal bacteria powder, which can be delivered to the intestines by oral administration, which can greatly reduce the impact of the digestive tract on the flora, and can significantly increase the rate of live bacteria to the intestines compared to encapsulation with acid-resistant capsules. Compared with other drug delivery technologies, sustained-release dripping pills can greatly improve the convenience of taking and improve patient compliance.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a multi-layer dripping pill encapsulating bioactive microorganisms, such as probiotics, intestinal flora, enzymes, polypeptides, etc., wherein a typical intestinal flora is a powder of intestinal flora obtained by removing impurities and freeze-drying feces provided by healthy volunteers. In particular, the dripping pills of the present invention, especially the multi-layer dripping pills containing intestinal flora, can be effectively used to treat certain mental illnesses such as autism spectrum disorders. The dripping pills of the present invention, especially the multi-layer dripping pills containing intestinal flora, can be called Huanglong dripping pills. Background Art

[0002] At present, there are many studies on the physical and chemical encapsulation and controlled release of bioactive substances (probiotics, intestinal flora) at home and abroad, and most of them use a single or two-layer aqueous phase system for encapsulation and controlled release. For example, polysaccharides such as sodium alginate and chitosan are used, and proteins such as soy protein and gelatin are used for encapsulation; there are also oil substances such as propolis, shellac, hardened oils, animal and plant oils; there are also technologies such as the use of two or more substances such as polysaccharide compounding, protein compounding, polysaccharide protein compounding, lipid and protein compounding; in addition, chemical modification is used to hydrophobically modify hydrophilic substances such as polysaccharides and proteins, and the speed of controlled release is controlled by controlling the degree of hydrophobic modification. Most of these technologies use hydrophilic systems to disperse bioactive substances (such as probiotics and intestinal flora), which has a great impact on their stability and vitality, greatly affecting the effect and yield, thereby increasing costs; some technical processes are relatively complex, with many steps and high industrialization difficulties; in addition, most of the single-layer single-phase and double-layer double-phase aqueous phase dispersion systems used are difficult to ensure that bioactive substances (such as probiotics) are not activated, thus affecting their stability. In addition, since bioactive substances are generally sensitive to gastric acid, the protective effect of the process on the active substances is limited.

[0003] Dropping pills are a high-end dosage form in the field of pharmacy. Due to their special structure, they can give special properties to medicines. Dropping pills are small pill-shaped preparations made by heating, melting and mixing solid or liquid drugs with appropriate substances (generally called matrix), dropping them into immiscible condensate, shrinking and condensing, and are mainly used for oral administration. From the composition and preparation method of the drop pill, it has the following characteristics: 1. Simple equipment, convenient operation, conducive to labor protection, short process cycle and high productivity; 2. The process conditions are easy to control, the quality is stable, the dosage is accurate, the heating time is short, and the stability of easily oxidized and volatile drugs can be increased after being dissolved in the matrix; 3. The matrix can accommodate a large amount of liquid drugs, so the liquid drugs can be solidified, such as the rue oil drop pills with an oil content of up to 83.5%; 4. The drop pills prepared by solid dispersion technology have the characteristics of rapid absorption and high bioavailability. For example, the effective dose of griseofulvin drop pills is 1 / 4 of 100 mesh fine powder and 1 / 2 of micro powder (particle size below 5 microns); 5. New dosage forms for ear and eye drugs have been developed. Otorhinolaryngology preparations are mostly liquid or semi-solid dosage forms, and the action time is not long-lasting. The drop pills can have a prolonged effect.

[0004] Drop pills have the advantage of "three effects", which refer to rapid effect, high efficiency and long-term effect. Drop pills are mostly taken sublingually. The drug is directly absorbed through the sublingual mucosa and enters the blood circulation, avoiding the first-pass effect of the liver caused by swallowing and the degradation loss of the drug in the stomach, so that the drug reaches the target organ at a high concentration and takes effect quickly. Generally, it will take effect within 5-15 minutes of sublingual administration, and no more than 30 minutes at most. Some also add sustained-release agents, which can significantly prolong the half-life of the drug and achieve the purpose of long-term effect. When needed, just hold it in your mouth.

[0005] At present, there are mainly the following types of drop pills: Rapid-acting and high-efficiency drop pills: Drop pills are prepared using solid dispersion technology. When the matrix dissolves, the drug in the body is released in the form of fine crystals, amorphous particles or molecules, so it dissolves quickly, absorbs quickly, acts quickly, and has high bioavailability. A typical example of this type of product is the rapid-acting heartache drop pill. Sustained-release and controlled-release drop pills: Sustained-release is to make the drug in the drop pill dissolve slowly over a long period of time to achieve long-term effect; controlled-release is to make the drug dissolve at a constant rate in the drop pill, and its effect can last for more than several days, such as chloramphenicol controlled-release eye pills. Solution drop pills: The lubricants and disintegrants used in tablets are mostly water-insoluble, so tablets are usually not used to prepare clear solutions. However, drop pills can be prepared with water-soluble matrices and can disintegrate into clear solutions in water, such as chloramphenicol drop pills, which can be used for drinking water disinfection. Suppository drop pills: Drop pills can use water-soluble matrices such as polyethylene glycol like water-soluble suppositories, and are dissolved by body fluids when used in cavities to produce effects. Such as norfloxacin ear drops, metronidazole tooth drops, etc. Drops can also be used in the rectum, and can also be absorbed by the rectum and act directly on the whole body. They have the characteristics of high bioavailability and fast action. Hard capsule drops: Drops with different solubility can be loaded into hard capsules to form sustained-release pellets with the required solubility, such as bifendate hard capsule drops. Coated drops: Like tablets and pills, they need to be coated with sugar coating, film coating, etc., such as bifendate drops. Liposome drops: Liposomes are suspension liquids, which can be made into solid dosage forms with polyethylene glycol. Liposomes are added to molten polyethylene glycol 4000 under continuous stirring to form a suspension, and then poured into a mold to condense and form. Enteric coated drops: Use a matrix that is insoluble in the stomach, such as potassium antimony tartrate drops, which are made into pills with gelatin solution as the matrix and then treated with formaldehyde to make the amino group of gelatin insoluble in gastric juice and soluble in the intestine. Dry-pressed coated pills: With the pill as the center, a coating composed of other drugs is pressed on it, which combines the advantages of two dosage forms, such as the antitussive and expectorant Kebiqing potassium chloride dry-pressed coated tablets. The former is the pill and the latter is the coating.

[0006] Classic dripping pills are not layered, and the dripping pills are a uniform mixture from the inside to the outside. With the development of preparations, there is a strong demand for the development of multi-layer dripping pills (such as three-layer dripping pills). The preparation process of these multi-layer dripping pills, such as double-layer dripping pills, is different from the soft capsules produced by the dripping method, because the contents of the soft capsules are liquid, while the internal components of the multi-layer dripping pills, such as three-layer dripping pills, are basically solid at room temperature. Therefore, the soft capsule production equipment / process cannot be applied to multi-layer dripping pills (such as three-layer dripping pills).

[0007] At present, the mechanical equipment for multi-layer drop pills is quite mature. For example, Chinese patent application No. 2016101875809 relates to multi-layer drop pill equipment and its components and the preparation process of multi-layer drop pills, Chinese patent application No. 2016108267433 relates to a method for improving the structure of multi-layer drop pills and the equipment used therein, and Chinese patent application No. 2019101573963 relates to a method for preparing high-uniformity drop pills and the equipment used therein, etc. These multi-layer drop pill equipments make it possible to produce multi-layer drop pills.

[0008] There are currently various compositions for supplementing the nutrition of both humans and animals, and these supplements can be provided to change, reduce or increase the microbial flora in the intestinal tract of an individual, thereby producing a desired effect on digestion. Ideally, supplementation can be based on changing the specific bacteria in the gastrointestinal (GI) tract of the human body to cultivate improved microbial flora for an individual (including humans). This type of supplementation can be carried out by using probiotics, which are understood to be surviving microorganisms, and when given in an effective amount, they give health or nutritional benefits to the host. Probiotics can provide a variety of benefits to the host, such as maintaining healthy gastrointestinal flora, enhancing immunity, preventing diarrhea, atopic dermatitis and other diseases, etc. The flora of the lower gastrointestinal tract, especially the colon part, plays an important role in maintaining the normal digestive tract function of the human body, but the problems caused by dysbacteriosis are also common and serious, such as constipation, diarrhea, etc. may be caused by dysbacteriosis. In recent years, there are also many literature reports that dysbacteriosis is related to certain mental illnesses such as autism, etc. It is believed that the administration of probiotics can restore normal flora in the gastrointestinal tract, which can be used to treat autism spectrum disorder (ASD) or autism. For example, see Ning Li et al. (Li N, et al. Fecal Microbiota Transplantation Relieves Gastrointestinal and Autism Symptoms by Improving the Gut Microbiota in an Open-Label Study. Front. Cell. Infect. Microbiol. 11: 759435. doi: 10.3389 / fcimb.2021.759435, and see Dae-Wook Kang et al. (Dae-Wook Kang, et al. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome (2017) 5: 10 DOI 10.1186 / s40168-016-0225-7).

[0009] Autism spectrum disorder (ASD) is a serious developmental disorder, a neurodevelopmental disorder characterized by social interaction disorders, verbal and nonverbal communication disorders, narrow interests, and stereotyped repetitive behaviors. It has been reported that in the United States, the prevalence of ASD in children and adolescents is 1 in 59, or even as high as 1%, and has been increasing in recent years. To date, the cause of ASD is still unclear, and there is no good treatment measure, and education and rehabilitation training are still the main treatment. In recent years, studies have found that ASD may be related to intestinal flora imbalance. Intestinal flora imbalance can cause a series of gastrointestinal symptoms and abnormal behaviors, and fecal microbiota transplantation can rebuild the patient's intestinal flora, relieve their gastrointestinal symptoms, improve some behavioral problems, and has a good therapeutic effect. Fecal microbiota transplantation is to transplant the functional flora in the feces of healthy people into the gastrointestinal tract of patients to rebuild new intestinal flora and achieve the treatment of intestinal and extraintestinal diseases. Traditional fecal microbiota transplantation mainly includes upper gastrointestinal enema, lower gastrointestinal enema, and oral fecal microbiota capsules. However, both the upper and lower gastrointestinal routes are complicated to operate, require the assistance of instruments in the hospital, and have certain risks. It is not convenient for repeated transplantation. Relatively speaking, oral fecal microbiota capsules are a more convenient and safe transplantation method. Most clinical fecal microbiota capsules are double-layer hydroxypropyl methylcellulose (HPMC) acid-resistant capsules. The smallest No. 3 capsule is 13.6mm long and 5.83mm in outer diameter. Although double-layer capsules are used, they are not completely sealed, and fecal microbes will stick to the capsule shell. Fecal microbiota capsules still have a noticeable odor and poor oral compliance. Most children with autism develop the disease at the age of 2-3 years old. The earlier the intervention treatment, the better the effect. However, for children with autism who are mostly younger, it is more difficult to swallow fecal microbiota capsules.

[0010] There is a very strong practical need to deliver live microorganisms such as probiotics or intestinal flora to the human gastrointestinal tract. Improving the human digestive tract flora and thus improving the gastrointestinal function of the human body as a whole is what technicians in this field are eagerly looking forward to. Summary of the invention

[0011] The purpose of the present application is to provide a method for delivering live microorganisms such as probiotics or intestinal flora directly to the gastrointestinal tract. Another purpose of the present application is to prepare a multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) structured drop pill for realizing the method. It has been unexpectedly found that the multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) drop pill having the structural features of the present application can beneficially deliver the expected amount of live microorganisms such as probiotics or intestinal flora directly to the gastrointestinal tract. The present application is completed based on this discovery.

[0012] To this end, the first aspect of the present invention provides a method for delivering live microorganisms such as probiotics or intestinal flora directly to the gastrointestinal tract, which is by giving a person in need thereof a multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dripping pills encapsulating live probiotics. Alternatively, the first aspect of the present invention provides the use of multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dripping pills in the preparation of a nutritional composition for delivering live microorganisms such as probiotics or intestinal flora directly to the gastrointestinal tract. The method of the first aspect below also relates to this use.

[0013] According to the method of the first aspect of the present invention, the multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pills include a multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) structure of (a) an inner layer, (b) a middle layer, and (c) an outer layer present in concentric circles, wherein:

[0014] (a) The inner layer is a homogeneous mixture of live microorganisms such as probiotics or intestinal flora powder and oily substances;

[0015] (b) The middle layer is oily;

[0016] (c) The outer layer is a rubber shell formed by dripping and drying a glue solution including a polymer material, a plasticizer and water.

[0017] According to the method of the first aspect of the present invention, the live microorganisms such as probiotics or intestinal flora are live microorganisms that are beneficial to the gastrointestinal tract.

[0018] According to the method of the first aspect of the present invention, the live microorganisms such as probiotics or intestinal flora can be in the form of their commercial sources or can be in the form of intestinal flora such as feces collected from healthy volunteers after impurity treatment or can be in the form of culture and expansion. Usually, the live microorganisms such as probiotics or intestinal flora used to prepare the multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dripping pills of the present invention are added in a powder state.

[0019] According to the method of the first aspect of the present invention, the live probiotics are selected from the genera Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Propionibacterium, Pediococcus, Staphylococcus, Bacillus, and Kluyveromyces.

[0020] According to the method of the first aspect of the present invention, the live probiotics are selected from the following live probiotics of the genus Bifidobacterium:

[0021] Bifidobacterium adolescentis, Bifidobacterium animalis such as Bb-12, Bifidobacterium lactis such as HN019 or Bi-07, Bifidobacterium bifidum, Bifidobacterium breve such as M-16V, Bifidobacterium infantis, Bifidobacterium longum such as BB536.

[0022] According to the method of the first aspect of the present invention, the live probiotics are live probiotics selected from the following Lactobacillus genus: Lactobacillus acidophilus such as NCFM, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactis subsp. lactis, Lactobacillus fermentum such as CECT5716, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus sakei, and Lactobacillus curvature.

[0023] According to the method of the first aspect of the present invention, the live probiotics are live probiotics selected from the following Streptococcus, Lactococcus, Leuconostoc, or Propionibacterium: Streptococcus thermophilus, Lactococcus lactis lactis, Lactococcus lactis cremoris, Lactococcus lactis diacetyl, Leuconostoc mesenteroides mesenteroides, Propionibacterium freudenreichii subsp. schreberi, and Propionibacterium acidipropionici.

[0024] According to the method of the first aspect of the present invention, the live probiotics are live probiotics selected from the following genera: Pediococcus acidilactici, Staphylococcus suis, Staphylococcus xylosus, Staphylococcus carnosus, Bacillus coagulans.

[0025] According to the method of the first aspect of the present invention, the weight ratio of the inner layer: the middle layer: the outer layer is 1:1.2-4:0.5-3.

[0026] According to the method of the first aspect of the present invention, the weight ratio of the inner layer: the middle layer: the outer layer is 1:1.5-3.5:1-2.5.

[0027] According to the method of the first aspect of the present invention, the weight ratio of the inner layer: the middle layer: the outer layer is 1:2.5-3.5:1-2.

[0028] According to the method of the first aspect of the present invention, the weight ratio of the inner layer: the middle layer: the outer layer is 1:3:1.5.

[0029] According to the method of the first aspect of the present invention, the polymer material in the outer layer is selected from gelatin, agar, gellan gum, carrageenan, furcellaran, pectin, chitosan, alginic acid, curdlan, starch, modified starch, pullulan, mannan and combinations thereof. In one embodiment, the polymer material is gelatin.

[0030] According to the method of the first aspect of the present invention, the plasticizer in the outer layer is selected from glycerol, sorbitol, and combinations thereof. In one embodiment, the plasticizer is glycerol.

[0031] According to the method of the first aspect of the present invention, in the rubber solution used for dripping to form the outer layer of the rubber shell, the weight ratio of the polymer material, the plasticizer and water is 1:0.05-0.2:2-4.

[0032] According to the method of the first aspect of the present invention, in the glue liquid used for dripping to form the outer layer of the rubber shell, the weight ratio of the polymer material, the plasticizer and the water is 1: 0.05-0.15: 2.5-3.5. In the present invention, when describing the weight ratio of the inner layer: the middle layer: the outer layer, the weight of the outer layer refers to the weight after drying and removing water. When dripping, the weight ratio of the outer layer in multiple layers (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) can be converted by the proportion of the glue liquid ingredients.

[0033] According to the method of the first aspect of the present invention, the glue liquid used for dripping to form the outer layer of the rubber shell also contains sucrose, wherein the weight ratio of the polymer material to the sucrose is 1:0.1-0.5, for example 1:0.1-0.3.

[0034] According to the method of the first aspect of the present invention, the glue solution used for dripping to form the outer layer of the rubber shell contains gelatin, glycerol, sucrose and water, for example, in a weight ratio of 1:0.05-0.2:0.1-0.5:2-4, for example, 1:0.05-0.15:0.1-0.3:2.5-3.5.

[0035] According to the method of the first aspect of the present invention, the polymer material is gelatin, and the gelatin jelly strength is 160-200.

[0036] According to the method of the first aspect of the present invention, the oily substances in the inner layer (a) and the middle layer (b) are independently selected from: hardened oils (e.g., melting point 39-43°C), vegetable oils (e.g., melting point 37-42°C), edible vegetable oils, sucrose fatty acid esters (SAIB), glycerol fatty acid esters, transesterified oils of palm oil, transesterified oils of palm fractionated oils, palm stearin, palm olein, palm super olein, palm diolein and palm intermediate fractions obtained by fractionating palm oil, medium-chain fatty acid glycerides, cocoa butter, cocoa butter substitutes, and combinations thereof. Unless otherwise specified, the oils obtained by transesterification or fractionation processes used herein are non-hydrogenated oil esters. For example, the edible vegetable oils are selected from: palm oil, coconut oil, peanut oil, soybean oil, sesame oil, olive oil, sunflower seed oil, rapeseed oil, tea seed oil, corn oil, and combinations thereof. These oily substances can be easily purchased from commercial sources, such as from commercial channels such as Yihai Kerry, COFCO Group, and Indonesia Musim Gold.

[0037] According to the method of the first aspect of the present invention, the middle layer (b) contains an oily substance such as hardened fat (e.g., melting point 39-43° C.), natural vitamin E oil and calcium chloride. For example, the middle layer material contains 1-5% (e.g., 1.5-3%) natural vitamin E oil, 1-5% (e.g., 2.5-4%) calcium chloride and the remainder hardened fat.

[0038] According to the method of the first aspect of the present invention, the melting point of the oily substance in the inner layer (a) is 35-55°C, such as 35-50°C, such as 35-45°C, such as 37-42°C.

[0039] According to the method of the first aspect of the present invention, wherein (a) the inner layer is a mixture consisting of an oily substance and live microorganisms such as probiotics or intestinal flora in a powdered state. In one embodiment, the live microorganisms such as probiotics or intestinal flora in a powdered state account for 10 to 40% of the weight of the inner layer, and for example, the live microorganisms such as probiotics or intestinal flora in a powdered state account for 20 to 30% of the weight of the inner layer.

[0040] According to the method of the first aspect of the present invention, the inner layer (a) contains: 10-40% of biologically active substances, 1-3% of natural vitamin E oil, 0.5-2.5% of dipotassium hydrogen phosphate-potassium dihydrogen phosphate (1:5), and the remainder of vegetable oil (e.g., melting point 37-42°C); for example, the inner layer (a) contains: 20-30% of biologically active substances, 1.8-2.2% of natural vitamin E oil, 0.8-1.2% of dipotassium hydrogen phosphate-potassium dihydrogen phosphate (1:5), and the remainder of vegetable oil (e.g., melting point 37-42°C).

[0041] According to the method of the first aspect of the present invention, the melting point of the oily substance in the middle layer (b) is 35-55°C, such as 35-50°C, such as 37-45°C, such as 39-43°C.

[0042] According to the method of the first aspect of the present invention, the multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) pellets prepared therefrom have a diameter of 1-10 mm, e.g., 1-8 mm, e.g., 1-7 mm, e.g., 2-5 mm.

[0043] According to the method of the first aspect of the present invention, the multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dropping pills are prepared by a method comprising the following steps:

[0044] (1) Preparing the inner layer material: first, uniformly mix the inner layer material except for live microorganisms such as probiotics or bacterial powder of intestinal flora, and slowly heat to a temperature 8 to 12° C. higher than the melting point of the material while stirring, and stir until the material is completely melted, then add powdered live microorganisms such as probiotics or bacterial powder of intestinal flora while maintaining the material at this temperature, continue stirring to uniformly mix the bacterial powder, obtain a homogeneous inner layer material, and keep it warm for dripping into dripping pills;

[0045] (2) preparing the middle layer material: mixing the materials uniformly, slowly heating them to a temperature 10 to 15° C. higher than the melting point of the materials while stirring, stirring until the materials are completely melted to obtain a homogeneous middle layer material, and keeping the temperature to prepare dripping pills;

[0046] (3) Preparing the outer layer material: soaking the polymer material in an appropriate amount of water to dissolve it, adding a plasticizer (and sucrose) to dissolve it, and adding water to the full amount to obtain the outer layer material, which is kept at a temperature 15 to 20° C. higher than the melting point of the middle layer material to prepare dripping pills;

[0047] (4) On a multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pill equipment, the inner layer material, the middle layer material, and the outer layer material are respectively dropped from the inner, middle, and outer layers (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) of a multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) structure dripper (e.g., the dripper temperature is set to be higher than 60° C.) into a cooling liquid (e.g., liquid paraffin) at 18 to 20° C. to cool and form multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) concentric structure seamless dropping pills, and then the obtained multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pills are dried by forced air at 15° C. to 20° C. for 10 to 12 hours to remove moisture from the outer layer, thereby obtaining multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pills.

[0048] Furthermore, the second aspect of the present invention provides a multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dropping pill, comprising a multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) structure of (a) an inner layer, (b) a middle layer, and (c) an outer layer present in concentric circles, wherein:

[0049] (a) The inner layer is a homogeneous mixture of live microorganisms such as probiotics or intestinal flora powder and oily substances;

[0050] (b) The middle layer is oily;

[0051] (c) The outer layer is a rubber shell formed by dripping and drying a glue solution including a polymer material, a plasticizer and water.

[0052] The multi-layer (eg, 2-5 layers, eg, 2-4 layers, eg, 3 layers) dropping pills according to the second aspect of the present invention have various technical features or combinations of technical features as described above in the first aspect.

[0053] Furthermore, the third aspect of the present invention provides a method for preparing the multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dropping pills according to any one of the second aspects of the present invention, comprising the following steps:

[0054] (1) Preparing the inner layer material: first, uniformly mix the inner layer material except for live microorganisms such as probiotics or bacterial powder of intestinal flora, and slowly heat to a temperature 8 to 12° C. higher than the melting point of the material while stirring, and stir until the material is completely melted, then add powdered live microorganisms such as probiotics or bacterial powder of intestinal flora while maintaining the material at this temperature, continue stirring to uniformly mix the bacterial powder, obtain a homogeneous inner layer material, and keep it warm for dripping into dripping pills;

[0055] (2) preparing the middle layer material: mixing the materials uniformly, slowly heating them to a temperature 10 to 15° C. higher than the melting point of the materials while stirring, stirring until the materials are completely melted to obtain a homogeneous middle layer material, and keeping the temperature to prepare dripping pills;

[0056] (3) Preparing the outer layer material: soaking the polymer material in an appropriate amount of water to dissolve it, adding a plasticizer (and sucrose) to dissolve it, and adding water to the full amount to obtain the outer layer material, which is kept at a temperature 15 to 20° C. higher than the melting point of the middle layer material to prepare dripping pills;

[0057] (4) On a multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pill equipment, the inner layer material, the middle layer material, and the outer layer material are respectively dropped from the inner, middle, and outer layers (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) of a multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) structure dripper (e.g., the dripper temperature is set to be higher than 60° C.) into a cooling liquid (e.g., liquid paraffin) at 18 to 20° C. to cool and form multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) concentric structure seamless dropping pills, and then the obtained multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pills are dried by forced air at 15° C. to 20° C. for 10 to 12 hours to remove moisture from the outer layer, thereby obtaining multi-layer (e.g., 2-5 layers, e.g., 2-4 layers, e.g., 3 layers) dropping pills.

[0058] Furthermore, the fourth aspect of the present invention provides the use of the multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) dropping pills as described in any one of the second aspects of the present invention in the preparation of drugs for treating certain mental illnesses such as autism spectrum disorders.

[0059] Alternatively, the fourth aspect of the present invention provides a method for treating certain mental illnesses such as autism spectrum disorders, which comprises administering to a subject in need thereof a therapeutically effective amount of the multi-layer (e.g., 2 to 5 layers, e.g., 2 to 4 layers, e.g., 3 layers) drops of the invention described in any one of the second aspects of the present invention.

[0060] In the steps of the above-mentioned preparation method of the present application, although the specific steps described therein are different from the steps described in the preparation examples in the specific implementation mode section below in some details or in the language description, those skilled in the art can fully summarize the above-mentioned method steps based on the detailed disclosure of the full text of the present application.

[0061] Any embodiment of any aspect of the present application can be combined with other embodiments, as long as they do not conflict. In addition, in any embodiment of any aspect of the present application, any technical feature can be applied to the technical features in other embodiments, as long as they do not conflict.

[0062] The present application is further described below.

[0063] All documents cited in this application are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with those in this application, the meanings expressed in this application shall prevail. In addition, the various terms and phrases used in this application have general meanings known to those skilled in the art, but even so, this application still hopes to provide more detailed descriptions and explanations of these terms and phrases, and if the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in this application shall prevail.

[0064] The term "microorganism" herein refers to a microorganism with low or no pathogenicity that exerts a beneficial effect on the health of the host. "Live microorganism" refers to a living or active microorganism that exerts a beneficial effect on the health of the host.

[0065] The term "probiotic" herein refers to microorganisms with low or no pathogenicity that exert a beneficial effect on the health of the host. "Live probiotic" refers to live or active microorganisms that exert a beneficial effect on the health of the host.

[0066] The term "intestinal flora" herein refers to microorganisms with low or no pathogenicity that play a beneficial role in the health of the host. "Living intestinal flora" refers to living or active microorganisms that play a beneficial role in the health of the host. Preferably, the bacterial powder of the intestinal flora is obtained from or provided from the feces of healthy volunteers after being treated by methods such as removing impurities and drying.

[0067] The term "sustained release" herein refers to the dropping pills prepared by the method of the present invention, which can regulate the release of bioactive substances to specific parts of the gastrointestinal tract, such as targeted release to the intestine.

[0068] The multilayer sustained-release drop pills provided by the present invention are used to encapsulate biologically active substances, especially for encapsulating live probiotics or fecal bacteria powder (which can be called Huanglong drop pills at this time), which can be delivered to the intestines by oral means, which can greatly reduce the impact of the digestive tract on the flora, and can significantly increase the rate of live bacteria to the intestines compared to encapsulating with acid-resistant capsules. Compared with other drug delivery technologies, sustained-release drop pills can greatly improve the convenience of taking and improve patient compliance. The fecal bacteria multilayer drop pills provided by the present invention can be mixed with the inner wall material for microencapsulation and then embedded, and are protected by a water-proof middle layer matrix and an oxygen-proof outer layer matrix. The finished product is a nearly spherical drop pill with a particle size of 1-10 mm, and by adding edible pigments and flavors, the color and taste of the fecal bacteria themselves are changed, which is easy to swallow and has good compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 : Flow chart of the general preparation method of three-layer dropping pills. DETAILED DESCRIPTION

[0070] The present application can be further described by the following examples, however, the scope of the present application is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present application without departing from the spirit and scope of the present application. The present application provides a general and / or specific description of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present application are well known in the art, the present application is still described as detailed as possible herein. The following examples further illustrate the present application, rather than limiting the present application. In the following specific examples, the formula is represented by a ratio of parts by weight, and when preparing the composition, the amount of each batch of feed is not less than 500g in total.

[0071] General preparation method of materials:

[0072] In the present invention, unless otherwise specified, various materials are prepared by conventional operations in combination with their properties.

[0073] For example, for the inner layer of a three-layer dropping pill, first mix the inner layer materials except the biologically active substance evenly, and slowly heat to a temperature 8 to 12° C. higher than the melting point of the material while stirring, and stir until the material is completely melted. Then, add the powdered biologically active substance while maintaining the material at this temperature, and continue stirring to evenly mix the bacterial powder to obtain a homogeneous inner layer material, which is then kept warm for making dropping pills.

[0074] For another example, for the middle layer of a three-layer dropping pill, the materials are mixed evenly and slowly heated to a temperature 10-15° C. higher than the melting point of the materials while stirring, and stirred until the materials are completely melted to obtain a homogeneous middle layer material, which is then kept warm for making dropping pills.

[0075] For another example, for the outer layer of a three-layer dropping pill, the polymer material is soaked in a proper amount of water to dissolve, other materials are added to dissolve, and water is added to the full amount to obtain the outer layer material, which is kept warm at a temperature 15 to 20°C higher than the melting point of the middle layer material to prepare the dropping pills.

[0076] General method of making three-layer dropping pills:

[0077] Using the existing three-layer pellet equipment (such as disclosed in Chinese Patent Application No. 2016101875809, Chinese Patent Application No. 2016108267433, Chinese Patent Application No. 2019101573963, etc.), the inner layer material, the middle layer material and the outer layer material are made from the inner layer material recorded in Chinese Patent Application No. 2019101573963. Figure 1 The dripper with a structure (the dripper temperature is 65°C) is dripped into a coolant (liquid paraffin) at 18-20°C to cool and form a three-layer concentric structure seamless pill (for convenience, a dripper of appropriate size is used to drip a three-layer pill with a diameter of 1-10 mm), and then the obtained three-layer pill is dried by forced air at 15°C-20°C for 10-12 hours to obtain a three-layer pill, which is then sealed and stored in a dark place at a temperature of 2-8°C. For a general preparation method flow chart of the three-layer pill, see Figure 1 .

[0078] Method for preparing intestinal flora powder, namely fecal bacteria powder

[0079] (1) Donor screening: Physiological indicators are mainly completed through scientific measurement and laboratory testing. ① Age 18-30 years old, body mass index (BMI) 18.5-23.9 kg / m2; ② Hematological examination: normal blood routine, liver and kidney function, electrolytes, and C-reactive protein, and negative tests for hepatitis virus, HIV, syphilis, Epstein-Barr virus, cytomegalovirus, COVID-19 antibodies, nematodes, and amebas; ③ Stool test: normal stool routine examination, negative occult blood test, negative tests for Clostridium difficile, Campylobacter, Salmonella, Shigella, Shiga toxin-producing Escherichia coli and eggs, vesicles, parasites, Enterobacter sakazakii, Yersinia enterocolitica, pathogenic Vibrio (Vibrio parahaemolyticus, Vibrio cholerae), Aeromonas, amebas, spores, norovirus, rotavirus, and new coronavirus (COVID-19), negative tests for multidrug resistance genes (extended-spectrum β-lactamase, carbapenemase, and vancomycin-resistant, etc.), and negative stool Helicobacter pylori antigen. Psychological indicators are mainly completed through interviews and questionnaires. ① The psychological state is good as determined by the psychologist or counselor; ② The scores of the self-rating depression scale (SDS), self-rating anxiety scale (SAS), Pittsburgh sleep quality index (PSQI), etc. are normal. Personal situation: mainly completed through interviews and questionnaires. ① Past history: no gastrointestinal discomfort in the past 2 weeks, no use of antibiotics, acid suppressants, immunosuppressants, chemotherapy drugs, blood transfusion history, etc. in the past 3 months, no chronic pain symptoms, no history of digestive system surgery, no history of infectious diseases or contact with infectious diseases, no history of allergic diseases, autoimmune diseases, metabolic diseases, cardiovascular and cerebrovascular diseases, nervous system or mental diseases, no history of malignant tumors, no intravenous injections of growth hormone, insulin, coagulation factors, etc.; ② Personal history: regular work and rest, healthy diet, harmonious family, no bad sexual intercourse, no smoking, drinking, drug abuse and other hobbies, no drug addiction, no vaccination or drug trial in the past 6 months, no tattoo or skin damage in the past 6 months, no travel history in tropical areas in the past 6 months; ③ Family history: no family history of gastrointestinal lesions, no family history of malignant tumors, no family history of infectious diseases; ④ Others: non-pregnant, non-menstrual period. Genetic indicators are completed by gene spectrum testing. Donors with positive pathogenic genes for single-gene hereditary diseases (especially recessive inheritance) should be excluded.

[0080] (2) Preparation of fecal powder: Use sterile containers for collection. The weight of each feces collection should be no less than 100g. The feces should be qualified only if the properties are 3-5 points in the Bristol scoring standard. Immediately enter the bacterial solution preparation process, or immediately seal and store at 2-8℃; Mix the feces with sterile saline in a ratio of 1:3, stir thoroughly and filter, and ensure that the time from feces excretion to completion of bacterial solution preparation is within 2 hours. The entire processing should be performed under a sterile environment; Place the bacterial solution in a freeze dryer, freeze-dry for 48 hours, then divide into packages and refrigerate at 4℃. If a donor meets the requirements, continuously collect, prepare and mix fecal powder for the preparation of pills.

[0081] Example 1: Three-layer dripping pills with high bioactive substances embedded

[0082] 1. Outer matrix

[0083] The outer layer matrix formula is: 23% gelatin (freezing strength 180), 2% glycerol, 5% sucrose, and water added to 100%. Preparation of the outer layer matrix: Soak the gelatin in an appropriate amount of water (overnight) to dissolve, add glycerol and sucrose, stir to dissolve, and add water to the full amount.

[0084] 2. Middle layer matrix

[0085] Middle layer matrix formula: hardened oil (melting point 39-43°C, measured 41°C) 95%, natural vitamin E oil 2%, calcium chloride 3%. Middle layer matrix preparation: mix all materials evenly and melt at 60°C, homogenize with a 5000r / min homogenizer to obtain.

[0086] 3. Inner capsule material

[0087] Inner capsule material formula: biologically active substances (such as probiotics, intestinal flora, enzymes, and polypeptides, added in an amount of 10-30%; in this example, the feces provided by healthy volunteer A was freeze-dried to obtain intestinal flora powder, and the added amount was 25%), natural vitamin E oil 2%, dipotassium hydrogen phosphate-potassium dihydrogen phosphate (1:5) 1%, vegetable oil (melting point 37-42°C, measured 39°C) added to 100% (added amount 60-80%, adjusted according to the added amount of biologically active substances). Inner capsule material preparation: melt the vegetable oil, add natural vitamin E oil and micronized dipotassium hydrogen phosphate-potassium dihydrogen phosphate, mix well, then add the biologically active substances, homogenize with a 5000r / min homogenizer, keep warm and stir for use to avoid sedimentation.

[0088] 4. Pill making

[0089] The weight ratio of the outer layer / middle layer / inner layer is 1:3:1.5. On the three-layer dropping pill equipment, the inner layer material, the middle layer material and the outer layer material are respectively dropped from the inner, middle and outer layers of the three-layer structure dripper (the dripper temperature is 65°C) into a cooling liquid (liquid paraffin) at 18-20°C to cool to form a three-layer concentric structure seamless dropping pill (the obtained dropping pill has a diameter of 2.5mm), and then the obtained three-layer dropping pill is air-dried at 15°C-20°C for 10-12 hours to remove the outer layer moisture to obtain a three-layer dropping pill, which is sealed and filled with ordinary gelatin hollow capsules, each with 400 mg, and stored in a dark place at a temperature of 2-8°C.

[0090] The obtained drop pills were tested to contain 5.7×10 7 cfu / g, active anaerobic bacteria 11.3×10 7 cfu / g, number of live bifidobacteria: 4.1×10 7 cfu / g pill (calculated based on feed amount and live bacteria count of bacterial powder).

[0091] Take the drop pill sample of Example 1, and carry out in simulated gastric juice and simulated intestinal juice according to "Test Example 1: Dissolution Determination Method" (3 capsules are placed in each dissolution cup), take samples at regular intervals, and then measure the survival rate of microorganisms in the sample after treatment with gastric juice or intestinal juice according to the method of "Test Example 2: Intestinal flora culture detection method". The results are shown in Table 1.

[0092] Table 1:

[0093]

[0094] The dripping pill sample is the sample of Example 1; the acid-resistant capsule is an acid-resistant hollow capsule (hydroxypropyl methylcellulose capsule, produced by Suzhou Capsule Co., Ltd.) sealed with intestinal flora powder, and the filling amount is consistent with the dripping pill capsule.

[0095] Next, the following three kinds of dropping pills were prepared with reference to Example 1 and their performances were investigated. Example 1a: Using intestinal flora powder from the same donor, referring to the formula and preparation method of Example 1, the only difference is that sucrose is not added to the outer layer matrix, and three-layer dropping pill #1a is obtained. Example 1b: Using intestinal flora powder from the same donor, referring to the formula and preparation method of Example 1, the only difference is that calcium chloride is not added to the middle layer matrix, and three-layer dropping pill #1b is obtained. Example 1c: Using intestinal flora powder from the same donor, referring to the formula and preparation method of Example 1, the only difference is that sucrose is not added to the outer layer matrix and calcium chloride is not added to the middle layer matrix, and three-layer dropping pill #1c is obtained. Referring to the determination method of the results in Table 1 of Example 1, the microbial survival rates of different fungi in different media in the three kinds of supplemented dropping pills were determined, and the results are shown in Table 2 below.

[0096] Table 2:

[0097]

[0098]

[0099] Example 2: Three-layer dripping pills with high bioactive substances embedded

[0100] 1. Outer matrix

[0101] The outer layer matrix formula is: 25% gelatin (freezing strength 160), 1.5% glycerol, 4% sucrose, and water added to 100%. Preparation of the outer layer matrix: Soak the gelatin in an appropriate amount of water (overnight) to dissolve, add glycerol and sucrose, stir to dissolve, and add water to the full amount.

[0102] 2. Middle layer matrix

[0103] Middle layer matrix formula: hardened oil (melting point 39-43°C, measured 43°C) 93%, natural vitamin E oil 3%, calcium chloride 4%. Middle layer matrix preparation: mix all materials evenly and melt at 60°C, homogenize with a 5000r / min homogenizer to obtain.

[0104] 3. Inner capsule material

[0105] Inner capsule material formula: biologically active substances (such as probiotics, intestinal flora, enzymes, and polypeptides, added in an amount of 10-30%; in this example, the intestinal flora powder obtained by removing impurities and freeze-drying the feces provided by healthy volunteer B, added in an amount of 20%), natural vitamin E oil 1.8%, dipotassium hydrogen phosphate-potassium dihydrogen phosphate (1:5) 1.2%, vegetable oil (melting point 37-42°C, measured 38°C) added to 100% (added amount 60-80%, adjusted according to the amount of biologically active substances added). Inner capsule material preparation: melt the vegetable oil, add natural vitamin E oil and micronized dipotassium hydrogen phosphate-potassium dihydrogen phosphate, mix well, then add the biologically active substances, homogenize with a 5000r / min homogenizer, keep warm and stir for use to avoid sedimentation.

[0106] 4. Pill making

[0107] The weight ratio of the outer layer / middle layer / inner layer is 1:3:1.5. On the three-layer dropping pill equipment, the inner layer material, the middle layer material and the outer layer material are respectively dropped from the inner, middle and outer layers of the three-layer structure dripper (the dripper temperature is 65°C) into a cooling liquid (liquid paraffin) at 18-20°C to cool to form a three-layer concentric structure seamless dropping pill (the obtained dropping pill has a diameter of 2.5mm), and then the obtained three-layer dropping pill is air-dried at 15°C-20°C for 10-12 hours to remove the outer layer moisture to obtain a three-layer dropping pill, which is sealed and filled with ordinary gelatin hollow capsules, each with 400 mg, and stored in a dark place at a temperature of 2-8°C.

[0108] The obtained drop pills were tested to contain 7.1×10 7 cfu / g, active anaerobic bacteria 9.7×10 7 cfu / g, number of live bifidobacteria: 3.8×10 7 cfu / g pill (calculated based on feed amount and live bacteria count of bacterial powder).

[0109] Referring to the determination method of the results in Table 1 of Example 1, the microbial survival rates of different fungi in the three-layer dripping pills obtained in this example in different media were determined, and the results were:

[0110] For aerobic bacteria, the survival rate of the pills in gastric juice for 2 hours was 72.4%, and the survival rate of the acid-resistant capsules was 14.7%, and the survival rate of the pills in intestinal juice for 4 hours was 27.2%, and the survival rate of the acid-resistant capsules was 8.4%; for anaerobic bacteria, the survival rate of the pills in gastric juice for 2 hours was 64.3%, and the survival rate of the acid-resistant capsules was 10.7%, and the survival rate of the pills in intestinal juice for 4 hours was 29.8%, and the survival rate of the acid-resistant capsules was 6.4%; for bifidobacteria, the survival rate of the pills in gastric juice for 2 hours was 69.7%, and the survival rate of the acid-resistant capsules was 20.4%, and the survival rate of the pills in intestinal juice for 4 hours was 36.4%, and the survival rate of the acid-resistant capsules was 14.7%.

[0111] The above results are slightly different from those in Example 1, but the difference is not obvious. This difference may be caused by different bacterial flora powder donors and / or slight differences in formulations.

[0112] Next, the following three kinds of dropping pills were supplemented and prepared with reference to Example 2 and their performances were examined. Example 2a: Using intestinal flora powder from the same donor, referring to the formula and preparation method of Example 2, the only difference is that sucrose is not added to the outer layer matrix, and three-layer dropping pill #2a is obtained. Example 2b: Using intestinal flora powder from the same donor, referring to the formula and preparation method of Example 2, the only difference is that calcium chloride is not added to the middle layer matrix, and three-layer dropping pill #2b is obtained. Example 2c: Using intestinal flora powder from the same donor, referring to the formula and preparation method of Example 2, the only difference is that sucrose is not added to the outer layer matrix and calcium chloride is not added to the middle layer matrix, and three-layer dropping pill #2c is obtained. Referring to the determination method of the results in Table 1 of Example 1, the microbial survival rates of different fungi in different media in the three kinds of supplemented dropping pills were determined, and the results are shown in the following Table 3.

[0113] Table 3:

[0114]

[0115] Example 3: Three-layer dripping pills with high bioactive substances embedded

[0116] 1. Outer matrix

[0117] The outer layer matrix formula is: 21% gelatin (freeze strength 200), 2.5% glycerol, 6% sucrose, and water added to 100%. Preparation of the outer layer matrix: Soak the gelatin in an appropriate amount of water (overnight) to dissolve, add glycerol and sucrose, stir to dissolve, and add water to the full amount.

[0118] 2. Middle layer matrix

[0119] Middle layer matrix formula: hardened oil (melting point 39-43°C, measured 39°C) 96%, natural vitamin E oil 1.5%, calcium chloride 2.5%. Middle layer matrix preparation: mix all materials evenly and melt at 60°C, homogenize with a 5000r / min homogenizer to obtain.

[0120] 3. Inner capsule material

[0121] Inner capsule material formula: biologically active substances (such as probiotics, intestinal flora, enzymes, polypeptides, added in an amount of 10-30%; this embodiment is commercially available probiotics, namely bifidobacterium powder, added in an amount of 30%), natural vitamin E oil 2.2%, dipotassium hydrogen phosphate-potassium dihydrogen phosphate (1:5) 0.8%, vegetable oil (melting point 37-42°C, measured 41°C) added to 100% (added amount 60-80%, adjusted according to the amount of biologically active substances added). Inner capsule material preparation: melt the vegetable oil, add natural vitamin E oil and micronized dipotassium hydrogen phosphate-potassium dihydrogen phosphate, mix well, then add the biologically active substances, homogenize with a 5000r / min homogenizer, keep warm and stir for use to avoid sedimentation.

[0122] 4. Pill making

[0123] The weight ratio of the outer layer / middle layer / inner layer is 1:3:1.5. On the three-layer dropping pill equipment, the inner layer material, the middle layer material and the outer layer material are respectively dropped from the inner, middle and outer layers of the three-layer structure dripper (the dripper temperature is 65°C) into a cooling liquid (liquid paraffin) at 18-20°C to cool to form a three-layer concentric structure seamless dropping pill (the obtained dropping pill has a diameter of 2.5mm), and then the obtained three-layer dropping pill is air-dried at 15°C-20°C for 10-12 hours to remove the outer layer moisture to obtain a three-layer dropping pill, which is sealed and filled with ordinary gelatin hollow capsules, each with 400 mg, and stored in a dark place at a temperature of 2-8°C.

[0124] It was determined that the number of live probiotics contained in the obtained pellets was 860 million cfu / g pellet (calculated based on the feed amount and the number of live bacteria in the bacterial powder).

[0125] The pellet samples of Example 3 were taken and tested in simulated gastric juice and simulated intestinal juice according to "Test Example 1: Dissolution Determination Method" (3 capsules were placed in each dissolution cup), and samples were taken at regular intervals. Then, according to the method of "Test Example 3: Probiotic Viable Bacteria Count Detection Method", the survival rate of microorganisms in the samples after being treated with gastric juice or intestinal juice was determined. The results are shown in Table 4.

[0126] Table 4:

[0127] Test items Pill survival rate Acid-resistant capsule survival rate 0h 100％ 100％ Simulated gastric fluid 2h 74.1％ 1.4％ Simulated intestinal fluid 4h 21.5％ 0.31％

[0128] The dripping pill sample is the sample of Example 3; the acid-resistant capsule is a bifidobacterium powder sealed with an acid-resistant hollow capsule, and the filling amount is consistent with the dripping pill capsule.

[0129] Next, the following three kinds of dropping pills were supplemented and prepared with reference to Example 3 and their performances were investigated. Example 3a: Using the same batch of bifidobacterium powder, referring to the formula and preparation method of Example 3, the only difference was that sucrose was not added to the outer layer matrix, and three-layer dropping pill #3a was obtained. Example 3b: Using the same batch of bifidobacterium powder, referring to the formula and preparation method of Example 3, the only difference was that calcium chloride was not added to the middle layer matrix, and three-layer dropping pill #3b was obtained. Example 3c: Using the same batch of bifidobacterium powder, referring to the formula and preparation method of Example 3, the only difference was that sucrose was not added to the outer layer matrix and calcium chloride was not added to the middle layer matrix, and three-layer dropping pill #3c was obtained. Referring to the determination method of the results in Table 4 of Example 3, the microbial survival rates of bifidobacteria in different media in the three supplemented dropping pills were determined, and the results are shown in Table 5.

[0130] Table 5:

[0131]

[0132]

[0133] According to the results of the above-mentioned Examples 1 to 3, it is shown that it is beneficial to add sucrose to the outer layer matrix and calcium chloride to the middle layer matrix.

[0134] Example 4: Three-layer dripping pills with high bioactive substances embedded

[0135] With reference to Example 1, the following three kinds of dropping pills were prepared and their performances were investigated. Example 4x: Using the bacterial powder of intestinal flora obtained by freeze-drying the feces provided by healthy volunteer X, referring to the formula and preparation method of Example 1, three-layer dropping pill #4x was obtained. Example 4y: Using the bacterial powder of intestinal flora obtained by freeze-drying the feces provided by healthy volunteer Y, referring to the formula and preparation method of Example 1, three-layer dropping pill #4y was obtained. Example 4z: Using the bacterial powder of intestinal flora obtained by freeze-drying the feces provided by healthy volunteer Z, referring to the formula and preparation method of Example 1, three-layer dropping pill #4z was obtained.

[0136] The results showed that the three kinds of pellets contained 4.6-8.7×10 7 cfu / g, active anaerobic bacteria 7.3~13.4×10 7 cfu / g, number of live bifidobacteria: 2.6~5.4×10 7 cfu / g pill (calculated based on feed amount and live bacteria count of bacterial powder).

[0137] Referring to the determination method of the results in Table 1 of Example 1, the microbial survival rates of different fungi in the three supplementary prepared dropping pills in different media were determined. The results are shown in the following Table 6.

[0138] Table 6:

[0139]

[0140] By comparing the survival rate of probiotics and intestinal flora dripping pills with multi-layer coating and double-layer acid-resistant capsules in artificial gastric juice for 2 hours and artificial intestinal juice for 4 hours, it can be seen that the multi-layer coated dripping pills are significantly better than the double-layer acid-resistant capsules. In addition, through data analysis, the multi-layer coated dripping pills can effectively prevent gastric acid from damaging bioactive substances and play a significant protective role.

[0141] Test Example 1: Dissolution Determination Method

[0142] The third method (small cup method) of "0931 Dissolution and Release Determination Method" in Part IV of the 2020 edition of the Chinese Pharmacopoeia was used to perform a dissolution test with 200 ml of dissolution medium, at a rotation speed of 50 rpm. Samples were taken after 2 hours of simulated gastric fluid or 4 hours of simulated intestinal fluid. According to the method of "Test Example 3: Probiotic Live Bacteria Count Detection Method", the survival rate of the microorganisms of the test samples after dissolution treatment was determined (the relative survival rate was calculated as 100% without dissolution treatment, i.e., the microbial survival rate at 0 h, and was expressed as the average of 6 dissolution cups).

[0143] The preparation method of dissolution medium simulated gastric fluid is as follows: take 3.84 ml of hydrochloric acid, add about 800 ml of water and 10 g of pepsin, shake well, dilute with water to 1000 ml, and obtain the obtained product. The preparation method of dissolution medium simulated intestinal fluid is as follows: take 6.8 g of potassium dihydrogen phosphate, add 500 ml of water to dissolve, adjust the pH value to 6.8 with 0.1 mol / L sodium hydroxide solution; take 10 g of pancreatic enzyme, add appropriate amount of water to dissolve, mix the two liquids, and dilute with water to 1000 ml, and obtain the obtained product.

[0144] Test Example 2: Intestinal flora culture detection method

[0145] 1. Buffer: 0.9% NaCl solution containing 0.05% tween-80 (sterilized in advance).

[0146] 2. Culture medium: ① Plate count agar (PCA) (HB0101, Haibo Biotechnology Co., Ltd.); ② Wilkins-Chalgren agar (WCA) (HB0261, Haibo Biotechnology Co., Ltd.); ③ MRS culture medium (027315, Guangdong Huankai Microbiological Technology Co., Ltd.), and the supporting reagents SR0370 mupirocin lithium salt and cysteine ​​hydrochloride need to be added according to the instructions.

[0147] 3. Preparation of enterobacteria pellet solution: 0.5 g pellets were added to 49.5 g sterilized buffer and homogenized at 18000 rpm for 80 s;

[0148] 4. Enterobacteria pellets test: (When determining the number of live bacteria in the pellets) the prepared Huanglong pellet solution was diluted with buffer gradient to the appropriate concentration and then applied: PCA was used to culture total aerobic bacteria, and the culture was carried out at 37°C after application; WCA was used to culture total anaerobic bacteria, and the culture was carried out at 37°C after application; MRA was used to culture bifidobacteria, and the culture was carried out at 37°C after application. Plate counts were performed after 48 hours.

[0149] For dissolution testing, the solution is directly coated and incubated.

[0150] 5. Calculation of results

[0151] A. If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the average of the colony counts on the two plates and then divide the average by the corresponding dilution gradient to obtain the total number of colonies per gram of sample.

[0152] B. If the colony counts on two serial dilution plates are within the appropriate counting range, calculate according to formula ①:

[0153]

[0154] Where: N is the number of colonies in the sample; ∑C is the sum of the number of colonies on the plate (containing a plate with a suitable range of colony numbers); n 1 is the number of plates of the first dilution (low dilution multiple); n 2 is the number of plates for the second dilution (high dilution multiple); d is the dilution factor (first dilution).

[0155] Test Example 3: Probiotic viable bacteria count detection method

[0156] 1. Prepare 198 mL of anaerobic dilution solution, sterilize at 121°C for 15 minutes, and cool to room temperature for later use.

[0157] 2. In the clean bench, use a sterilized measuring cylinder to take 198 mL of sterile anaerobic dilution solution, pour it into a sterile conical flask, and then keep it warm at 37°C.

[0158] 3. (When determining the number of viable bacteria in the pills) Randomly select 2 g of sample, put it into a conical flask containing 198 ml of diluent, and then keep it at 37°C for 7 minutes.

[0159] 4. Homogenize at a speed of 18,000 rpm for 7 minutes to fully release the probiotics in the pills.

[0160] 5. Use a 1 ml sterile pipette to quickly take 1 ml of the homogenized sample in the clean bench, add it to 9 ml of anaerobic diluent, and mix it with a vortex mixer to prepare 1×10 -1 diluent.

[0161] 6. Take another 1mL sterile pipette tip and perform 10-fold incremental dilution according to the method in step 5. Replace the sterile pipette tip once for each incremental dilution.

[0162] 7. Pipette 1 mL of appropriate gradient dilution solution into sterile plates, and prepare two plates for each dilution. After the dilution solution is transferred into the plate, pour about 15 mL of RCM agar medium cooled to 48°C into the plate, and rotate the plate to mix evenly. Incubate anaerobic culture at 36°C±1°C for 48h±2h24h±2h. The process from sample dilution to plate pouring should be completed within 15 minutes.

[0163] For the dissolution test, 1 mL of the dissolution solution was directly pipetted into a sterile plate and the test was performed in the same manner.

[0164] 8. The method for counting live bacteria shall be implemented in accordance with 6.3 of GB4789.35. After the culture is completed, select the plates with colony counts between 30CFU and 300CFU and no spreading colony growth to count the total number of colonies. The plates with less than 30CFU shall record the specific number of colonies, and those with more than 300CFU shall be recorded as too many to count. The number of colonies for each dilution shall be the average of two plates.

[0165] 9. Result calculation

[0166] (1) If only one dilution plate has a colony count within the appropriate counting range, calculate the average colony count on the two plates and divide the average by the corresponding dilution gradient to obtain the total colony count per gram of sample.

[0167] (2) If the colony counts on two consecutive dilution plates are within the appropriate counting range, calculate according to formula ①:

[0168]

[0169] Where: N is the number of colonies in the sample; ∑C is the sum of the number of colonies on the plate (containing a plate with a suitable range of colony numbers); n 1 is the number of plates of the first dilution (low dilution multiple); n 2is the number of plates for the second dilution (high dilution multiple); d is the dilution factor (first dilution).

[0170] Anaerobic dilution solution formula: 6.0g disodium hydrogen phosphate, 4.5g potassium dihydrogen phosphate, 0.8g Tween 80, 0.5g cysteine, 0.5g nutrient agar, 1000ml purified water. After complete dissolution, sterilize at 121℃ for 15 minutes.

[0171] RCM medium formula: agar 15.0g, peptone 10.0g, beef powder 10.0g, glucose 5.0g, sodium chloride 5.0g, yeast powder 3.0g, sodium acetate 3.0g, soluble starch 1.0g, L-cysteine ​​hydrochloride 0.5g, purified water 1000mL. After complete dissolution, sterilize at 121℃ for 15 minutes.

[0172] As described above, given that it is known that administration of intestinal flora from healthy volunteers can be effectively used to treat certain mental illnesses such as autism spectrum disorder (Autism spectrum disorder, ASD, commonly referred to as autism), the drop pills prepared by the present invention can be fully used for such therapeutic purposes.

[0173] Experimental Example 4: Autism Mouse Model Experiment

[0174] Autism mouse model experiments are well known in the art. This experimental example draws on relevant literature and conducts corresponding experiments in combination with the actual situation of the present invention.

[0175] The experiment used autism mouse model BTBR T+tf / J mice, 7-8 weeks old, weighing 25-30g. The control mice were C57BL / 6 mice, 7-8 weeks old, weighing 22-28g. All experimental animals were kept in the standard clean animal room of the Experimental Animal Center of the Army Medical University, with alternating day and night, constant temperature (18-22℃), and free and sufficient access to food and water.

[0176] To avoid the influence of litter size, male mice were used in the experiment, and the mice were divided into 3 groups (10 in each group) by random number table method: C57 control group, BTBR model group, and BTBR intervention group. Mice in the BTBR intervention group were all given Huanglong pills by gavage, and each animal was given 0.15g of the pills prepared in Example 1 by gavage once a day for a total of 7 days; the control group was given blank pills (equal weight) prepared by reference to Example 1 but without adding fecal bacteria.

[0177] 1. Open field test (OFT)

[0178] In 50×50cm 2The mice were recorded in a white plexiglass arena using an overhead camera, and the behavior of the mice was tracked and analyzed. Before testing, the arena was cleaned and disinfected with 75% alcohol and left vacant for at least 5 minutes to allow the odor to dissipate. The mice were then introduced into the arena and allowed to explore for 10 minutes while being tracked. The total distance traveled by the mice was analyzed, and the distance traveled was recorded in a 30×30 cm 2 The movement distance of the central area. The results are shown in Table 7 below.

[0179] Table 7:

[0180] Group Total movement distance / cm Central zone movement distance / cm The ratio of central area movement distance to total movement distance Rd C57 control group <![CDATA[2895.4±263.6 * ]]> 647.4±81.20 <![CDATA[0.22±0.04 * ]]> BTBR Model Group 3714.2±203.6 651±114.6 0.17±0.03 BTBR intervention group <![CDATA[3141.2±269.7 * ]]> 640.5±77.79 <![CDATA[0.20±0.03 * ]]>

[0181] Note: * represents p < 0.05, compared with the BTBR model group.

[0182] The results of the open field test showed that the total movement distance of the BTBR model group was significantly greater than that of the C57 control group, and the BTBR+Huanglong Pills intervention group was significantly improved compared with the BTBR model group. The proportion of central area movement distance to total movement distance in the BTBR model group was significantly lower than that in the C57 control group, and the BTBR+Huanglong Pills intervention group was significantly improved compared with the BTBR model group, and there was no significant difference with the C57 control group. The results showed that the anxiety behavior of the BTBR model group was more severe, and Huanglong Pills intervention could improve the anxiety level of BTBR mice.

[0183] 2. Three-chamber social experiment

[0184] The test mice were placed in a rectangular transparent social box (40cm×60cm×22cm) and allowed to move freely in three connected chambers for 10 minutes. The test mice were taken out and an empty cage and a cage with a stranger mouse 1 of the same species and age were randomly placed in the left and right chambers. The well-adapted test mice were gently placed in the middle chamber with their backs facing the experimenter and allowed to move around for 10 minutes. The time (T) for the test mice to interact with the empty cage and the stranger mouse 1 was recorded by video. E and T S ), and then gently remove the tested mouse. Place another stranger mouse 2 of the same species and age group in the empty cage, and then gently place the tested mouse in the middle chamber again. Record the time (T) for the tested mouse to interact with the previous stranger mouse and the new stranger mouse in the next 10 minutes. S1 and T S2 ). The results of the three-chamber social experiment are shown in Table 8 below.

[0185] Table 8:

[0186] Group C57 control group BTBR Model Group BTBR+Huanglong dripping pills intervention group <![CDATA[T E / s]]> 80.64±11.3 88.81±9.55 82.93±7.28 <![CDATA[T S / s]]> <![CDATA[116.92±14.1 * ]]> 79.98±9.03* 87.33±7.53 <![CDATA[T S1 / s]]> 88.51±5.45 78.21±5.63 82.26±5.61 <![CDATA[T S2 / s]]> <![CDATA[112.28±9.61 △ ]]> <![CDATA[75.96±4.29 △ ]]> <![CDATA[87.59±6.26 △ ]]>

[0187] Note: * represents p < 0.05, and T EComparison; △ represents p < 0.05, compared with T S1 Compare.

[0188] From the results of the three-chamber social experiment, we can see that the C57 control group T S Significantly greater than T E , T S2 Significantly greater than T S1 , indicating that the social ability and social novelty ability of C57 control group mice were normal. BTBR model group, T S Less than T E , T S2 ≈T S1 , indicating that the mice in the BTBR model group had social ability disorders and social novelty defects. S Greater than T E , T S2 Significantly greater than T S1 , indicating that Huanglong Pills intervention effectively improved the social disorder of BTBR mice.

[0189] 3. Buried bead test: It was carried out in a normal cage with the floor filled with 3-4 cm of fresh, autoclaved wood chip bedding. The mice were first adapted to the cage for 10 minutes and then transferred to another cage while the bedding was leveled and 20 glass marbles (4×5) were placed on it. The mice were then returned to their cages and taken out after 10 minutes, which were then recorded and photographed for reference. The bedding was changed for each mouse, and the glass beads were soaked in 75% ethanol and dried in the bedding between tests. When the experimenter was blinded to the treatment or group, the number of buried marbles was scored by a second experimenter who was blinded to the treatment / group from the second top-view image taken 10 minutes later. The results are shown in Table 9 below.

[0190] Table 9: Buried bead test

[0191] Group Number of buried beads C57 control group <![CDATA[9.1±1.91 * ]]> BTBR Model Group 15.4±1.71 BTBR+Huanglong dripping pills intervention group <![CDATA[12.8±1.81 * ]]>

[0192] Note: * represents p < 0.05, compared with BTBR group.

[0193] The results of the buried bead test showed that there was a significant difference between the BTBR group and the C57 control group (p < 0.01), and there was improvement compared with the BTBR + Huanglong Pills intervention group (p < 0.05), indicating that Huanglong Pills treatment can improve the abnormal repetitive stereotyped behavior of adult BTBR mice.

[0194] 4. Grooming behavior observation test

[0195] The test mice were placed in a 50cm×50cm×45cm test box for 10 minutes of adaptation, and then tested for 10 minutes. Two testers simultaneously recorded the number of times and total time the test mice combed their hair anywhere on their bodies. The third recorder counted the results. If the difference between the two recorded results in terms of grooming times was ≤5 and the difference in grooming time was ≤15s, the recorded test result was used. Otherwise, the third tester retested and took the average. The test area was kept quiet to avoid people walking around. When changing animals, the inner wall and bottom of the test box were wiped with 75% alcohol. The results of the grooming behavior observation test are shown in Table 10 below.

[0196] Table 10:

[0197] Group Grooming times Total grooming time / s C57 control group 14.7±3.40* <![CDATA[40.65±3.58 * ]]> BTBR Model Group 29.2±5.57 83.71±8.09 BTBR+Huanglong dripping pills intervention group <![CDATA[22.9±4.06 * ]]> <![CDATA[76.44±9.76 * ]]>

[0198] Note: * represents p < 0.05, compared with BTBR group.

[0199] The results of grooming times and total grooming time in the grooming experiment showed that there were significant differences between the BTBR group and the C57 control group (p < 0.01), and there was an improvement trend compared with the BTBR + Huanglong Drops intervention group (p < 0.05), indicating that Huanglong Drops treatment has a significant improvement effect on the abnormal repetitive stereotyped behavior of adult BTBR mice.

[0200] 5. New object recognition test

[0201] The test mice were adapted to the test room for 1 hour, and then the test mice were placed in the open field box (no object in the box) to adapt for 20 minutes. On the second day (after 24 hours of adaptation), the test mice were adapted to the test room for 1 hour, and then the test mice were placed in the open field box to adapt for at least 5 minutes, and then the test mice were taken out of the open field box and placed in a clean temporary cage for about 2 minutes. Two plastic triangles of the same size, color and texture were placed at the left and right ends of the same side in the open field box as familiar objects, and then the test mice were placed in the box with their backs facing the familiar objects, allowing them to explore freely for 10 minutes. After 30 minutes of familiarization period, a familiar object in the open field box was randomly replaced with a plastic pentagon of similar size and different colors as a novel object, and then the mouse was placed in the box with its back facing the object, and the exploration time (Tn) of the mouse to the novel object and the exploration time (Tf) of the familiar object were recorded in 5 minutes, and the timing standard was the time of exploration within 2 cm of the mouse nose or mouth from the object. The results are shown in Table 11 below.

[0202] Table 11:

[0203] Group C57 control group BTBR Model Group BTBR+Huanglong dripping pills intervention group Tf / s 12.26±1.24 16.46±1.79 15.84±1.17 Tn / s <![CDATA[17.21±1.058 * ]]> 17.71±1.580 18.82±1.026

[0204] Note: * represents p < 0.05, compared with Tf group.

[0205] In the novel object recognition test, Tn of the C57 control group was significantly greater than Tf, indicating that the C57 control group had normal memory. There was no significant difference between Tn and Tf in the BTBR model group, indicating that the mice had memory impairment. There was no significant difference between Tf and Tn in the BTBR+Huanglong Drop Pills intervention group, indicating that the mice still had memory impairment and the improvement was not obvious.

[0206] The above-described embodiments are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Equivalent substitutions or changes made by technicians in the technical field based on the present application are all within the protection scope of the present application. The protection scope of the present application shall be subject to the claims.

Claims

1. A multilayer dropping pill, comprising a three-layer structure of (a) an inner layer, (b) a middle layer, and (c) an outer layer in a concentric circle shape, wherein the weight ratio of the inner layer: the middle layer: the outer layer is 1:1.5-3.5:1-2.5, wherein: (a) the inner layer material comprises: 10-40% of biologically active substances, 1-3% of natural vitamin E oil, 0.5-2.5% of a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in a weight ratio of 1:5, and the remainder of vegetable oil with a melting point of 37-42° C., wherein the biologically active substances are live microbial probiotics or intestinal flora in a powdered state; (b) The middle layer material comprises: 1-5% natural vitamin E oil, 1-5% calcium chloride and the remainder hardened oil with a melting point of 39-43°C, (c) The outer layer is a rubber shell formed by dripping and drying a glue solution including polymer material gelatin, plasticizer glycerol, sucrose and water in a weight ratio of 1:0.05~0.2:0.1~0.5:2~4.

2. The multi-layered dropping pill according to claim 1, wherein the live microorganisms are probiotics or intestinal flora that are beneficial to the gastrointestinal tract.

3. The multi-layered dropping pill according to claim 1, wherein the live microorganism is a probiotic or intestinal flora, which is in the form of commercial sources or in the form of intestinal flora feces collected from healthy volunteers after impurity treatment or in the form of cultured and expanded.

4. The multi-layer dripping pill according to claim 1, wherein the probiotic is selected from the group consisting of Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Propionibacterium, Pediococcus, Staphylococcus, Bacillus, and Kluyveromyces.

5. The multi-layered dripping pill according to claim 4, wherein the Bifidobacterium is selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

6. The multi-layer dripping pill according to claim 4, wherein the Lactobacillus genus is selected from the group consisting of: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus sakei, and Lactobacillus curvature.

7. The multi-layer pellet according to claim 1, wherein: The weight ratio of the inner layer: middle layer: outer layer is 1:2.5~3.5:1~2.

8. The multi-layer pellet according to claim 1, wherein: The weight ratio of the inner layer, middle layer and outer layer is 1:3:1.

5.

9. The multi-layer pellet according to claim 1, wherein: The weight ratio of gelatin, glycerol, sucrose and water in the outer layer is 1:0.05~0.15:0.1~0.3:2.5~3.

5.

10. The multi-layer pellet according to claim 1, wherein: The freezing strength of gelatin is 160~200.

11. The multi-layer pellet according to claim 1, wherein: The middle layer material comprises 1.5-3% natural vitamin E oil, 2.5-4% calcium chloride and the balance of hardened oil with a melting point of 39-43°C.

12. The multi-layer pellet according to claim 1, wherein: The live microorganisms in the inner layer are a mixture of probiotics or intestinal flora.

13. The multi-layer pellet according to claim 1, wherein: The live microorganisms in powder form account for 20-30% of the weight of the inner layer.

14. The multi-layer pellet according to claim 1, wherein: The inner layer material comprises: 20-30% of biologically active substances, 1.8-2.2% of natural vitamin E oil, 0.8-1.2% of a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in a weight ratio of 1:5, and the remainder of vegetable oil with a melting point of 37-42°C. The multi-layer pellet according to claim 1 , having a diameter of 1 to 8 mm.

16. The multi-layer pellet according to claim 1, which has a diameter of 2 to 5 mm.

17. The multilayer pellet according to claim 1, which is prepared by a method comprising the following steps: (1) preparing the inner layer material: first, uniformly mix the inner layer materials except the powder of live microorganisms, and slowly heat to a temperature 8-12° C. higher than the melting point of the materials while stirring, and stir until the materials are completely melted, then add the powder of live microorganisms while maintaining the materials at this temperature, and continue stirring to uniformly mix the powder, to obtain a homogeneous inner layer material, and keep it warm for dripping into dripping pills; (2) preparing the middle layer material: mixing the materials uniformly, slowly heating them to a temperature 10-15° C. higher than the melting point of the materials while stirring, stirring until the materials are completely melted to obtain a homogeneous middle layer material, and keeping the temperature to prepare dripping pills; (3) preparing the outer layer material: soaking the polymer material in an appropriate amount of water to dissolve it, adding a plasticizer and sucrose to dissolve them, adding water to the full amount to obtain the outer layer material, and keeping it at a temperature 15 to 20° C. higher than the melting point of the middle layer material to prepare dripping pills; and (4) On a three-layer dropping pill device, the inner layer material, the middle layer material and the outer layer material are respectively dropped from the inner, middle and outer layers of the three-layer structure dripper with a temperature higher than 60° C. into a cooling liquid paraffin at 18 to 20° C. to form a three-layer concentric structure seamless dropping pill, and then the obtained three-layer dropping pill is dried by forced air at 15° C. to 20° C. for 10 to 12 hours to remove the outer layer moisture, thereby obtaining a three-layer dropping pill.

18. A method for preparing the multilayer dropping pills according to any one of claims 1 to 17, comprising the following steps: (1) preparing the inner layer material: first, uniformly mix the inner layer materials except the powder of live microorganisms, and slowly heat to a temperature 8-12° C. higher than the melting point of the materials while stirring, and stir until the materials are completely melted, then add the powder of live microorganisms while maintaining the materials at this temperature, and continue stirring to uniformly mix the powder, to obtain a homogeneous inner layer material, and keep it warm for dripping into dripping pills; (2) preparing the middle layer material: mixing the materials uniformly, slowly heating them to a temperature 10-15° C. higher than the melting point of the materials while stirring, stirring until the materials are completely melted to obtain a homogeneous middle layer material, and keeping the temperature to prepare dripping pills; (3) preparing the outer layer material: soaking the polymer material in an appropriate amount of water to dissolve it, adding a plasticizer and sucrose to dissolve them, adding water to the full amount to obtain the outer layer material, and keeping it at a temperature 15-20°C higher than the melting point of the middle layer material to prepare dripping pills; (4) On a three-layer dropping pill device, the inner layer material, the middle layer material and the outer layer material are respectively dropped from the inner, middle and outer layers of the three-layer structure dripper with a temperature higher than 60° C. into a cooling liquid paraffin at 18 to 20° C. to form a three-layer concentric structure seamless dropping pill, and then the obtained three-layer dropping pill is dried by forced air at 15° C. to 20° C. for 10 to 12 hours to remove the outer layer moisture, thereby obtaining a three-layer dropping pill.

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